TWI554284B - Pertuzumab variants and evaluation thereof - Google Patents

Description

Pertuzumab variant (PERTUZUMAB) and its evaluation

This non-provisional application filed under 37 CFR §1.53(b) asserts the US Provisional Application Serial Number filed on April 16, 2013 under 35 USC §119(e), which is cited in full by reference. Incorporated herein.

Sequence table

The invention includes a sequence listing submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy created on April 8, 2014 is named P5584R1-WO_Seq_Listing.txt and is 31,363 bits in size.

The present invention relates to a variant of Pertuzumab. In particular, the present invention relates to: an unpaired cysteine variant comprising one or two light chain variable domains of Cyst23/Cys88 unpaired cysteine, a Patuxin The afucosylated variant of the monoclonal antibody, a low molecular weight species of paltomizumab (LMWS), and a high molecular weight species of pattenzumab (HMWS). The invention further discloses isolated variants, compositions, pharmaceutical compositions and articles of manufacture comprising the variants, and methods of making and characterizing such variants and compositions thereof.

Pertuzumab (also known as rhuMAb 2C4) is a monoclonal antibody (MAb) that is the first of a series of agents known as "HER dimerization inhibitors". By binding to HER2, it inhibits the dimerization of HER2 with other HER receptors and thus inhibits tumor growth. Pertuzumab received US Food and Drug Administration on June 8, 2012 The Office (US FDA) approved for the treatment of HER2-positive metastatic breast cancer.

U.S. Patent No. 7,862,817 (Adams et al.) describes a humanized antibody to the 2C4 antibody, which is referred to as humanized 2C4 version 574 or recombinant humanized monoclonal antibody 2C4 (rhuMAb 2C4). This antibody binds to subdomain II in the extracellular domain (ECD) of human epidermal growth factor receptor 2 (HER2). The rhuMAb 2C4 anti-system was made on a laboratory scale and showed binding to HER2 and inhibited the growth of MDA-175 cells (which express HER2 at 1+ levels) and MCF7 xenografts implanted in mice. See also Adams et al, Cancer Immunol. Immunother . 55(6): 717-727 (2006).

U.S. Patent No. 6,339,142 (Blank and Base) describes a HER2 antibody composition comprising a mixture of an anti-HER2 antibody and one or more acidic variants thereof, wherein the (iso) acidic variant is present in an amount less than about 25%. Humanized monoclonal antibody 4D5 variant 8 (humMAb4D5-8 or trastuzumab) is an exemplary HER2 antibody.

U.S. Patent No. 7,560,111, U.S. Patent No. 7,879,325, and U.S. Patent No. 8,241,630 (Kao et al.) describe a variant of pertuzumab (rhuMAb 2C4) comprising one or two light chains of the antibody The amine-based terminal leader region extends (VHS-), the so-called "VHS-variant". When using the Elman analysis (Ellman's analysis) to test the reference material (Phase I), Lot S9802A (Phase II), and the free thiol of the 400L scale process development material under natural conditions, free sulfur in all tested materials The alcohol level is below the limit of detection. 1 to 2% of the pertuzumab in the composition tested was afucosylated (G0-F) as measured by capillary electrophoresis (CE). See Table 5 of U.S. Patent No. 7,560,111 (Kao et al.).

WO 2009/099829 (Harris et al.) describes acidic variants of pertuzumab comprising: deamination variants, glycosylation variants, disulfide reduction variants, non-reducible variants and sialylation Variants. These variants are characterized according to the disclosure as follows:

CEX = cation exchange. CE-SDS = capillary electrophoresis using sodium lauryl sulfate.

In WO 2009/099829 (Harris et al.), the experimental method for characterizing disulfide-reduction variants (non-reducing CE-SDS of intact antibodies) evaluates reduced interchain disulfide bonds rather than intrachain disulfide bonds.

Zhang et al, Anal. Chem. 84(16): 7112-7123 (2012) report a recombinant antibody (mAb A) with an unpaired cysteine (Cys22 and Cys96) in its variable heavy domain (VH domain) . These unpaired cysteines were found to have no significant effect on the binding of the antibody to CD20, and mAb A with unpaired cysteine was fully active in the potency assay (complement dependent cytotoxicity (CDC) assay).

WO 2009/009523 (Kao et al.) discloses inhibition of intrachain disulfide reduction during recombinant production of an opreclizumab (rhuMAb 2H7) antibody that binds to CD20.

Harris, R. Dev . Biol. (Basel, Switzerland) 122: 117-127 (2005) revealed in the variable weight (VH) domain of omalizumab, a humanized anti-IgE antibody Unpaired cysteine (Cys22 and Cys96). This unpaired cysteine form has a significantly reduced potency.

The experimental data herein relates to variant forms of pertuzumab which include unpaired cysteine variants, afucosylated variants, low molecular weight species (LMWS) and high molecular weight species (HMWS). Methods for identifying, characterizing, and quantifying such variants are of value in the manufacture and quality control methods for pertuzumab pharmaceutical compositions.

Thus, in a first aspect, the invention relates to a composition comprising pertuzumab and an unpaired cysteine variant thereof, wherein the unpaired cysteine variant comprises pertuzumab Cys23/Cys88 unpaired cysteine in one or both of the light chain variable domains. The unpaired cysteine variant includes a heterodimeric variant (including Cys23/Cys88 unpaired cysteine in only one light chain variable domain of pertuzumab) and/or homologous Polymer variants (including Cys23/Cys88 unpaired cysteine in the two light chain variable domains of pertuzumab).

The composition further includes one or more other variants of pertuzumab, such as afucosylation-free variants, low molecular weight species (LMWS) variants, high molecular weight species (HMWS) variants, saccharification Body, disulfide bond reduction variant, non-reducible variant, deamination variant, sialylation variant, VHS-variant, C-terminal lysine variant, methionine oxidative variant, G1 glycosylation variants, G2 glycosylation variants, and non-glycosylated heavy chain variants.

The invention also relates to a composition comprising a fucosylated variant of pertuzumab and pertuzumab, wherein the afucosylated variant is present in an amount from 0.9 to 4.1 of the composition %. In one embodiment, the invention is also directed to a composition comprising a fucosylated variant of pertuzumab and pertuzumab, wherein the afucosylated variant is greater than 2% of the composition. According to this embodiment, the amount of the afucosylated variant is greater than that reported in U.S. Patent No. 7,560,111, U.S. Patent No. 7,879,325, and U.S. Patent No. 8,241,630 (Kao et al.).

In another aspect, the present invention is directed to a composition comprising a mixture of low molecular weight species (LMWS) of pertuzumab, pertuzumab, and high molecular weight species (HMWS) of pertuzumab, The content of LMWS 1.6% and HMWS content 1.7%.

The invention also relates to a composition comprising a mixture of pertuzumab, peak 1 and peak 2, wherein the content of peak 1 is The content of 0.5% and peak 2 is 1.0% as measured by reductive capillary electrophoresis of sodium lauryl sulfate (R-CE-SDS) assay.

Other aspects of the invention pertain to pharmaceutical compositions, articles of manufacture, and methods of treating cancer patients using or comprising the compositions herein.

In another aspect, the invention relates to a method of evaluating a pertuzumab composition comprising: (1) measuring the content of an unpaired cysteine variant in the composition, wherein the unpaired The cysteine variant comprises Cys23/Cys88 unpaired cysteine in one or two light chain variable domains of pertuzumab; and/or (2) measuring fucose in the composition The content of the basest pertuzumab; and/or (3) the content of the low molecular weight species (LMWS) or high molecular weight species (HMWS) of the pertuzumab in the composition.

In still another aspect, the present invention is directed to a method of assessing the biological activity of a pertuzumab composition comprising measuring the amount of afucosylated betuzumab variant in the composition. The antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the composition was determined and confirmed to be in the range of from about 0.9% to about 4.1% of the fucosylated betuzumab.

In another aspect, the invention relates to a method of making a composition comprising: (1) preparing a composition comprising pertuzumab and one or more variants thereof, and (2) making the resulting The composition is subjected to an analytical assay to assess the content of the (etc.) variant therein, wherein the (etc.) variant comprises: (i) one or two light chain variable domains comprising pertuzumab An unpaired cysteine variant of Cys23/Cys88 unpaired cysteine; and/or (ii) an afucosylated variant of pertuzumab; and/or (iii) Patuxin High molecular weight species of monoclonal antibody (HMWS); and/or (iv) low molecular weight species of paltoizumab (LMWS), and/or (v) peak 1 fragment of pertuzumab and/or (vi) Peak 2 of Pertuzumab.

In another aspect, the invention relates to an isolated pertuzumab variant, wherein The isolated variant comprises: (a) an unpaired cysteine variant of pertuzumab, wherein the variant is Cys23/ in only one light chain variable domain comprising pertuzumab a heterodimeric variant of Cys88 unpaired cysteine; and/or (b) an unpaired cysteine variant of pertuzumab, wherein the variant is two comprising pertuzumab a homodimeric variant of Cys23/Cys88 unpaired cysteine in a light chain variable domain; and/or (c) an afucosylated variant of pertuzumab; and/or (d) high molecular weight species of palutizumab (HMWS); and/or (e) low molecular weight species of paltoizumab (LMWS); and/or (f) peak 1 fragment of pertuzumab And/or (g) a peak 2 fragment of pertuzumab.

In another aspect, the invention relates to a method of assessing fragmentation of a pertuzumab composition comprising measuring the combination by reductive capillary electrophoresis sodium dodecyl sulfate (R-CE-SDS) assay The content of peak 1 and peak 2 in the substance and the content of the peak 1 are confirmed to be 5% and the content of the peak 2 is 1.0%.

Figure 1 provides a schematic representation of the HER2 protein structure, and the amino acid sequences of subdomains I to IV of its extracellular domain (SEQ ID No. 1 to 4, respectively).

2A and 2B show the amino acid sequence alignment of the following: variable light (VL) (Fig. 2A) and variable weight (VH) (Fig. 2B) domains of mouse monoclonal antibody 2C4 (SEQ ID: SEQ ID, respectively) No. 5 and 6); variant 574 / VL and VH domains of pertuzumab (SEQ ID No. 7 and 8, respectively), and human VL and VH consensus framework (hum κ1, light κ subgroup I; humIII, baryon group III) (SEQ ID No. 9 and 10, respectively). The asterisk identifies the difference between the partial domain of pertuzumab and the murine monoclonal antibody 2C4 or between the pertuzumab and the variable domain of the human framework. The complementarity determining regions (CDRs) are in brackets.

Figures 3A and 3B show the amino acid sequence of the pertuzumab light chain (Figure 3A; SEQ ID NO. 11) and the heavy chain (Figure 3B; SEQ ID No. 12). The CDR system is shown in bold. The calculated molecular weights of the light and heavy chains are 23,526.22 Da and 49,216.56 Da (in reduced form of cysteine). The carbohydrate moiety is attached to the heavy chain Asn 299.

Figures 4A and 4B show the amino acid sequences of the trastuzumab light chain (Figure 4A; SEQ ID NO. 13) and the heavy chain (Figure 4B; SEQ ID NO. 14), respectively. The boundaries of the light chain variable domain and the heavy chain variable domain are indicated by arrows.

Figures 5A and 5B show the light chain sequence of the pertuzumab variant (Figure 5A; SEQ ID NO. 15) and the heavy chain sequence of the pertuzumab variant (Figure 5B; SEQ ID NO. 16), respectively. .

Figure 6 depicts the structure of (main species) pertuzumab, including its four strands and 12 intrachain disulfide bonds, each of which includes Cys23/Cys88 in the light chain variable (VL) domain. Sulfur bond. The domains depicted are: VL = light chain variable domain; VH = heavy chain variable domain; CL = light chain constant domain; CH1 = heavy chain constant domain 1; CH2 = heavy chain constant domain 2; CH3 = heavy chain Constant domain 3.

Figure 7 shows a non-reduced (primary) tryptic peptide map of pertuzumab.

Figure 8 depicts tryptic peptide maps (full scale) of reduced and non-reduced pertuzumab.

Figure 9 depicts a tryptic peptide map of reduced and non-reduced pertuzumab (0 to 120 minutes).

Figure 10 depicts tryptic peptide maps of reduced and non-reduced pertuzumab (120 to 204 minutes).

Figure 11 depicts hydrophobic interaction chromatography (HIC) analysis of papacilumab digested with papain.

Figure 12 depicts HIC analysis (enlarged view) of papacilumin digested with papain.

Figure 13 depicts HIC analysis of intact pertuzumab. Shows the following peaks: homodimers of free thiols (free thiols on two light chains), free thiol heterodimers (free thiols on a light chain) and Wild type homodimer (main type antibody).

Figure 14 depicts the activity of pertuzumab (batch anti2C4907-2) in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

Figure 15 depicts the effect of G0-F levels on ADCC activity. The sample tested was a phase III Tocilizumab (G0-F = 2.2%) and stage I pertuzumab (G0-F = 0.8%).

Figure 16 depicts capillary electrophoresis analysis of N-linked oligosaccharides released from pertuzumab.

Figure 17 depicts capillary electrophoresis analysis (enlarged view) of N-linked oligosaccharides released from pertuzumab. Note: G1 oligosaccharides have two isomeric forms (labeled G1 and G1') in which a terminal galactose residue is attached to an alpha 1-6 branched chain or an alpha 1-3 branched chain.

Figure 18 depicts reverse phase-high performance liquid chromatography (RP-HPLC) for the separation of pertuzumab Fab and Fc (limited Lys-C digestion). A limited Lys-C digested pertuzumab treated with N-ethylmaleimide (NEM) and a limited Lys-C digested pertuzumab were shown.

Figure 19 depicts a peptide map demonstrating that the pertuzumab free thiol Fab contains free Cys23 and Cys88 at its light chain. The L2 peptide from the Fab containing free thiol was labeled by NEM and thus shifted in peptide mapping analysis.

Figure 20 is a schematic representation of major species or wild type IgGl (Figure 20A), Cys23/Cys88 heterodimer variant (Figure 20B) and Cys23/Cys88 homodimer variant (Figure 20C).

Figure 21 depicts the G0-F% versus ADCC activity of the pertuzumab batch using the assay of Example 4 herein.

Figure 22 is a schematic representation of the binding of pertuzumab at the heterodimerization binding site of HER2, thereby preventing heterodimerization with activated EGFR or HER3.

Figure 23 compares the activity of trastuzumab (which binds to subdomain IV near the membrane domain of HER2 ECD) and pertuzumab (which binds to subdomain II of HER2 ECD).

Figures 24A and 24B depict oligosaccharide structures attached to IgG antibodies.

Figure 25 depicts size exclusion chromatography (SEC) analysis (full scale) of pertuzumab.

Figure 26 depicts SEC analysis (amplification scale) of pertuzumab. The peak includes a main peak (main type antibody), a high molecular weight species (HMWS), and a low molecular weight species (LMWS).

Figure 27 depicts size exclusion-high performance liquid chromatography (SE-HPLC) analysis of a pertuzumab sample. Sample A is a representative batch of representative pertuzumab drugs. Sample B accepts 1.2 mlux Batch of pastuzumab exposed to light. Sample C was a batch of pertuzumab that received 3.6 mlux of light exposure. Sample D was a batch of pertuzumab that was acid treated at pH 3.2. Sample E was a purified alkaline variant from ion exchange-HPLC (IE-HPLC).

Figure 28 is a graph showing the index of analytical ultracentrifugation (AUC) sedimentation velocity and SE-HPLC analysis. Error bars represent the two standard deviations from n = 3 measurements. All other data points represent a single determination. Circles indicate that the sample has a HMWS level below the detection level of AUC.

Figure 29 depicts capillary electrophoresis sodium dodecyl sulfate analysis (CE-SDS) using non-reduced pertuzumab using laser induced fluorescence (LIF) detection.

Figure 30 depicts CE-SDS-LIF (magnified view) of non-reducing (NR) pertuzumab.

31A and 31B are graphs showing the SE-HPLC chromatogram of Example 6: full scale (Fig. 31A) and enlarged scale (Fig. 31B).

32A and 32B are diagrams showing the non-reducing CE-SDS (NR-CE-SDS) electropherogram of Example 6: full scale (Fig. 32A) and enlarged scale (Fig. 32B).

33A and 33B are graphs showing the reduced CE-SDS (R-CE-SDS) electrophoresis of Example 6: full scale (Fig. 33A) and enlarged scale (Fig. 33B).

Figure 34 provides a comparison of NR-CE-SDS and R-CE-SDS electropherograms (magnification scales) of acid treated samples.

Figure 35 depicts the correlation of Fab quantification between NR-CE-SDS and SE-HPLC.

I. Definition

"Dopcysteine" as used herein refers to two cysteine residues that form a disulfide bond in a protein such as an antibody. The disulfide bonds may be interchain disulfide bonds (eg, disulfide bonds between the heavy and light chains of the antibody or between the two heavy chains of the antibody) or intrachain disulfide bonds (eg, in antibodies) Within the light chain or within the heavy chain of the antibody). Most IgGl antibodies include four interchain disulfide bonds and 12 intrachain disulfide bonds. See Figure 6.

"unpaired cysteine variants" are proteins (eg, antibodies, such as patezumab) A variant of the anti-), wherein one or more of the paired cysteines are not in a disulfide-bonded state. The unpaired cysteine may not be paired to form a disulfide bond (eg, when the protein initially folds into its tertiary structure) or may have formed a disulfide bond, but it subsequently breaks (eg, during manufacturing) Or when storing). Such unpaired cysteine acids are commonly referred to as free thiols or free sulfhydryl groups. In one embodiment, the unpaired cysteine acids are derived from intrachain disulfide bonds. In one embodiment, the unpaired cysteine is in the light chain of the antibody, such as the light chain variable domain. In one embodiment, the unpaired cysteine variant is a Cys23/Cys88 variant.

The "Cys23/Cys88" unpaired cysteine variant lacks an intramolecular disulfide bond at the cysteine residues 23 and 88 in one or both of the light chain variable domains of the antibody. See Figures 20(b) and (c) of this document.

"Homologous dimer variants" lack the Cys23/Cys88 disulfide bond in the two light chain variable domains of the antibody. See Figure 20(c) of this document.

A "heterodimeric variant" lacks only one Cys23/Cys88 disulfide bond in one of the light chain variable domains of the antibody. See Figure 20(b) of this document.

An afucosylated variant is a glycosylation variant of an antibody in which one or both of the oligosaccharide structures attached to one or both heavy chain residues Asn299 lack fucose, for example in Fucα (1->6) is absent in the core oligosaccharide structure.

The "low molecular weight species" or "LMWS" of pertuzumab comprises a fragment of pertuzumab which has a molecular weight less than the major species or intact pertuzumab (eg, wherein the complete pertuzumab has about The molecular weight of 145,197 Da, only the molecular weight of its peptide chain was measured. LMWS can be detected by size exclusion high performance liquid chromatography (SE-HPLC) and/or non-reducing capillary electrophoresis (CE-SDS) using sodium lauryl sulfate, for example as in Example 5. In one embodiment, the LMWS includes or consists of "Peak 6" (see, for example, Example 5) obtained by CE-SDS.

"High molecular weight species" or "HMWS" includes preparations of pertuzumab, which have The molecular weight is greater than the molecular weight of the major species or intact pertuzumab (eg, where the complete pertuzumab has a molecular weight of about 145,197 Da, only the peptide chain is measured). HMWS can be detected by size exclusion high performance liquid chromatography (SE-HPLC) and/or by non-reducing capillary electrophoresis (CE-SDS) using sodium lauryl sulfate, for example as in Example 5.

"Peak 1" herein refers to a fragment of a pertuzumab that is smaller in size than the pertuzumab light chain (LC). The peak 1 fragment can be isolated from the major class of pertuzumab by CE-SDS assay, preferably by reducing CE-SDS (R-CE-SDS) assay. See, for example, Figure 33B, Table 16, and Table 18 herein. Preferably, the content of peak 1 in the pertuzumab composition is 0.5%. The R-CE-SDS assay was performed as described in Example 6 and the corrected peak area (CPA) provided a peak of 1% in the composition, as appropriate.

"Peak 2" as used herein refers to a pertuzumab fragment having a size greater than the pertuzumab light chain (LC) and less than the pertuzumab non-glycosylated heavy chain (NGHC). Peak 2 can be isolated from the major class of pertuzumab by CE-SDS, preferably by reducing CE-SDS (R-CE-SDS) assay. Peak 2 does not include peak 3, which may occur during the R-CE-SDS assay as explained in Example 6 herein. See, for example, Figure 33B, Table 16, and Table 18 herein. Preferably, the content of peak 2 in the pertuzumab composition is 1.0%. The R-CE-SDS assay was performed as described in Example 6 and the corrected peak area (CPA) provided a peak of 2% in the composition, as appropriate.

"Fragmentation" refers to the cleavage of a polypeptide chain, for example, the cleavage of a light chain and/or heavy chain of a pertuzumab. It does not include, for example, dissociation of non-covalently associated polypeptide chains during NR-CE-SDS analysis.

An "analytical assay" is a assay that qualitatively assesses and/or quantifies the presence or amount of an analyte (eg, an antibody variant) in a composition. The composition that accepts the assay can be a purified composition, including a pharmaceutical composition.

"Fab hydrophobic interaction chromatography" or "Fab HIC assay" includes (eg, using papain) to produce fragments (eg, Fab fragments) of the antibody in the composition and to thereby produce The native antibody fragment received HIC to isolate the unpaired cysteine variant from the major class of pertuzumab. An exemplary assay is disclosed in Example 1 herein.

"HER receptor" is a receptor protein tyrosine kinase that belongs to the HER receptor family and includes the EGFR, HER2, HER3 and HER4 receptors. The HER receptor will generally comprise an extracellular domain that can bind to the HER ligand and/or dimerize with another HER receptor molecule; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and with several The carboxyl terminus signal domain of the phosphorylated tyrosine residue.

The expression "HER2" refers to the human HER2 protein described in, for example, Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al., Nature 319:230-234 (1986) (Genebank Accession No. X03363) ).

As used herein, "HER2 extracellular domain" or "HER2 ECD" refers to the domain of HER2 that is affixed to the cell membrane or circulated outside of the cell, including fragments thereof. The amino acid sequence of HER2 is shown in Figure 1. In one embodiment, the extracellular domain of HER2 can comprise four domains: "Subdomain I" (amino acid residues from about 1 to 195; SEQ ID NO: 1), "Subdomain II" (amino acid residues) From about 196 to 319; SEQ ID NO: 2), "Subdomain III" (amino acid residues from about 320 to 488: SEQ ID NO: 3) and "Subdomain IV" (amino acid residues from Between 489 and 630; SEQ ID NO: 4) (residue numbering of no signal peptide). See Garrett et al, Mol. Cell. 11:495-505 (2003), Cho et al, Nature 421:756-760 (2003), Franklin et al, Cancer Cell 5:317-328 (2004) and Plowman et al. , Proc. Natl. Acad. Sci. 90: 1746-1750 (1993), and Figure 1 herein.

As used herein, "HER dimer" is a non-covalently associated dimer comprising at least two HER receptors. Such complexes can be formed upon exposure of cells expressing two or more HER receptors to a HER ligand and can be isolated by immunoprecipitation and analyzed by SDS-PAGE, such as, for example, Sliwkowski et al. Human, J. Biol. Chem. , 269(20): 14661-14665 (1994). Other proteins, such as cytokine receptor subunits (eg, gp130), can associate with the dimer. Preferably, the HER dimer comprises HER2.

As used herein, "HER heterodimer" is a heterodimer comprising a non-covalent association of at least two different HER receptors, such as EGFR-HER2, HER2-HER3 or HER2-HER4 heterologous Dimer.

"HER activation" refers to the activation or phosphorylation of any one or more of the HER receptors. In general, HER activation results in signal transduction (eg, signal transduction by phosphorylation of a tyrosine residue in a HER receptor or substrate polypeptide by the intracellular kinase domain of the HER receptor). HER activation can be mediated by binding to a HER ligand comprising a HER dimer of the HER receptor of interest. A HER ligand that binds to a HER dimer can activate a kinase domain of one or more HER receptors in the dimer and thereby result in phosphorylation of the tyrosine residue of the one or more HER receptors and/or Or phosphorylation of other substrate polypeptides, such as tyrosine residues in Akt or MAPK intracellular kinases.

A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody comprising a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized anti-systems in which residues from the hypervariable region of the recipient are derived from non-human species such as mice, rats, rabbits or non-human spirits with the required specificity, affinity and ability Human immunoglobulin (recipient antibody) substituted with a residue of a hypervariable region (donor antibody) of a long animal. In some cases, the framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to their non-human immunoglobulins and all or substantially all of These are the FR human immunoglobulin sequences. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically one of the human immunoglobulins. For further details, see Jones et al , Nature 321 :522-525 (1986); Riechmann et al , Nature 332:323-329 (1988); and Presta, Curr.Op.Struct.Biol. 2:593-596 ( 1992). Humanized HER2 antibodies specifically include trastuzumab and humanized 2C4 antibodies, such as pertuzumab as described and defined herein.

As used herein, "intact antibody" refers to an antibody comprising two antigen binding regions and one Fc region. Preferably, the intact antibody has a functional Fc region. In one embodiment, "complete pertuzumab" has a molecular weight of about 145,197 Da, and only its peptide chain is measured.

As used herein, the term "hypervariable region" refers to an amino acid residue in an antibody that is responsible for antigen binding. The hypervariable region typically comprises amino acid residues from the "complementarity determining region" or "CDR" (eg, residues 24 to 34 (L1), 50 to 56 (L2), and 89 to 97 (in the light chain variable domain). L3) and 31 to 35 (H1), 50 to 65 (H2) and 95 to 102 (H3) in the heavy chain variable domain; Kabat et al ., Sequences of Proteins of Immunological Interest , 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or their residues from the "hypervariable loop" (eg residues 26 to 32 (L1), 50 to 52 (L2) in the light chain variable domain And 91 to 96 (L3) and heavy chain variable domains 26 to 32 (H1), 53 to 55 (H2) and 96 to 101 (H3); Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)). "Framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, which includes the original sequence Fc region and the variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as being unfolded from an amino acid residue at position Cys226, or from Pro230 to its carboxy terminus. The C-terminal acyl acid of the Fc region (according to the EU numbering system, residue 449) can be removed, for example, during preparation or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Thus, a composition of intact antibodies can comprise a population of antibodies that remove all K449 residues, a population of antibodies that do not have a K449 residue removed, and a population of antibodies that have a mixture of antibodies with or without K449 residues.

Unless otherwise indicated, the numbering of residues in the immunoglobulin heavy chain herein is numbered by the EU index, such as Kabat et al ., Sequences of Proteins of Immunological Interest , 5th edition, Public Health Service, National Institutes of Health. , Bethesda, MD (1991), which is expressly incorporated herein by reference. "EU index as Kabat" refers to the residue number of a human IgG1 EU antibody.

The "functional Fc region" has the "effector function" of the original sequence Fc region. Exemplary "effector functions" include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptor; BCR) ) Under the adjustment. Such effector functions generally require that the Fc region bind to a binding domain (eg, an antibody variable domain) and can be assessed using a variety of assays.

The "original sequence Fc region" includes the same amino acid sequence as the amino acid sequence of the Fc region found in nature. The original sequence human Fc region includes the original sequence human IgG1 Fc region (non-A and A allotype); the original sequence human IgG2 Fc region; the original sequence human IgG3 Fc region; and the original sequence human IgG4 Fc region, as well as naturally occurring variants thereof .

"Naked antibody" refers to an antibody that does not bind to a heterologous molecule, such as a cytotoxic moiety or a radioactive label.

The term "primary antibody" or "wild-type antibody" as used herein refers to the structure of an antibody amino acid sequence in a composition which is a quantitatively dominant antibody molecule in the composition. Preferably, the major class of antibodies are HER2 antibodies, such as antibodies that bind to subdomain II of HER2, antibodies that inhibit HER dimerization more efficiently than trastuzumab, and/or heterodimer binding sites that bind to HER2. Point antibody. In one embodiment, the major class anti-system comprises the following antibodies: CDR-H1 (SEQ ID NO: 17 or 23), CDR-H2 (SEQ ID NO: 18), and CDR-H3 (SEQ ID NO: 19) , CDR-L1 (SEQ ID NO: 20), CDR-L2 (SEQ ID NO: 21 or 24) and CDR-L3 (SEQ ID NO: 22), VL and VH in SEQ ID NO. 7 and 8, respectively An amino acid sequence (see Figures 2A to 2B) and, optionally, a light chain amino acid sequence of SEQ ID NO. 11 or 15 and a heavy chain amino acid sequence of SEQ ID NO. 12 or 16 (see 3A to 3B and 5A to 5B). In one embodiment, the major class of antibodies is pertuzumab.

An antibody that "inhibits HER dimerization" is an antibody that inhibits or interferes with the formation of a HER dimer or a heterodimer. In one embodiment, the antibody binds to HER2 at its heterodimeric binding site. The best dimerization inhibitory antibody herein is pertuzumab.

"Heterinary dimer binding site" on HER2 means that in the extracellular domain of HER2, when it forms a dimer with EGFR, HER3 or HER4, it contacts a region of the extracellular domain of EGFR, HER3 or HER4 or interacts with it. a district. This region was found to be in subdomain II of HER2 (SEQ ID NO: 2). Franklin et al, Cancer Cell 5:317-328 (2004).

a HER2 antibody that binds to a heterodimeric binding site of HER2, binds to a residue in subdomain II (SEQ ID NO: 2) and optionally binds to other domains of the HER2 extracellular domain (eg, a subdomain) Residues in I and III (SEQ ID NOS: 1 and 3)) and may sterically hinder (at least to some extent) the formation of HER2-EGFR, HER-HER3, or HER2-HER4 heterodimers . Franklin et al, Cancer Cell 5:317-328 (2004) characterize the crystal structure of HER2-Pertuzumab, deposited in the RCSB Protein Library (ID code 1S78), showing heterodimerization of HER2 binding to HER2 Exemplary antibodies to the body binding site.

An antibody that binds to subdomain II of HER2 binds to subdomain II of HER2 (SEQ ID NO: 2) and optionally other subdomains, such as subdomains I and III (SEQ ID NOS: 1 and 3, respectively). Residues.

For the purposes of this document, "partuxumab" and "rhuMAb 2C4" are used interchangeably to refer to a variable light chain (VL) and variable heavy chain (VH) comprising SEQ ID NOS: 7 and 8, respectively. An antibody to an amino acid sequence. Figures 22 and 23 herein illustrate exemplary biological functions of pertuzumab. Where pertuzumab is an intact antibody, it preferably comprises an IgGl antibody; in one embodiment, it comprises the light chain amino acid sequence of SEQ ID NO: 11 or 15, and SEQ ID NO: 12 or A heavy chain amino acid sequence of 16. The anti-system is prepared by recombining Chinese hamster ovary (CHO) cells as appropriate. The terms "paltuzumab" and "rhuMAb 2C4" herein encompass a biosimilar or pseudocopy of the drug having the US name (USAN) or International Nonproprietary Name (INN): Pertuzumab.

For the purposes of this document, "trastuzumab" and "rhuMAb4D5" are used interchangeably to refer to a variable light chain (VL) and variable heavy chain (VH) amine comprising SEQ ID Nos: 13 and 14, respectively. An antibody to the acid sequence (see Figures 4A to 4B). Where trastuzumab is an intact antibody, it preferably comprises an IgGl antibody; in one embodiment, it comprises the light chain amino acid sequence of SEQ ID NO: 13 and the heavy chain amine of SEQ ID NO: Base acid sequence. The antibody is optionally prepared by Chinese hamster ovary (CHO) cells. The terms "trastuzumab" and "rhuMAb4D5" herein encompass a biosimilar or pseudocopy of the drug having the US name (USAN) or the international non-proprietary name (INN): trastuzumab.

The amino acid sequence variant antibody herein is an antibody having an amino acid sequence different from the main class of antibodies. Typically, the amino acid sequence variant will have at least about 70% homology to the major class of antibodies, and preferably will be at least about 80%, more preferably at least about 90% homologous to the major class of antibodies. . Amino acid sequence variants have substitutions, deletions, and/or additions at or near certain positions in the amino acid sequence of the major class of antibodies. Examples of amino acid sequence variants herein include deamidated antibody variants, antibodies having an amine terminal leader extension (eg, VHS-) on one or both of its light chains, in one or both An antibody having a C-terminal amino acid residue on the heavy chain or the like, and a combination of amino acid sequence changes of the heavy chain and/or the light chain.

Acidic variants are variants of the major class of antibodies that are more acidic than the major classes of antibodies. Acidic variants have acquired a negative charge or a loss of positive charge relative to the major class of antibodies. Such acidic variants can be resolved using separation methods such as ion exchange chromatography, i.e., separation of proteins based on charge. The acidic variant of the major class of antibodies elutes earlier than the main peak when separated by cation exchange chromatography.

The disulfide bond reduction variant has one or more interchain disulfide linked cysteine acids that are chemically reduced to the free thiol form. This variant can be monitored by non-reducing capillary electrophoresis (CE-SDS) using sodium lauryl sulfate, for example as described in WO 2009/099829 (Harris et al.).

As used herein, a non-reducible variant or "incompletely reduced variant" is a variant of a major class of antibodies that cannot be chemically reduced to heavy and light chains by treatment with a reducing agent such as dithiothreitol. Such variants may be treated by treating the composition with a reducing agent and using methods for assessing the size of the protein, such as capillary electrophoresis (CE-SDS) using sodium lauryl sulfate, for example using WO2009/099829 (Harris et al). The techniques described were evaluated by evaluating the resulting composition.

A glycosylation variant antibody herein is an antibody having one or more carbohydrate moieties attached thereto that differ from one or more carbohydrate moieties attached to the major class of antibodies. In one embodiment, the glycosylation variant has an oligosaccharide structure attached to one or both heavy chains of the antibody, such as at residue 299 of the heavy chain. In one embodiment, the major class of antibodies (eg, pertuzumab) comprises a G0 oligosaccharide as the primary oligosaccharide attached to its Fc region. Exemplary oligosaccharide structures attached to IgGl are depicted in Figures 24A-24B. Examples of glycosylation variants herein include an afucosylated variant, an antibody having a G1 or G2 oligosaccharide structure but not a G0 oligosaccharide structure attached to its Fc region ("G1 glycosylation variant" Or a "G2 glycosylation variant"), an antibody that has no carbohydrate attached to one or both of the antibodies ("non-glycosylated heavy chain variant"), a sialylation variant, etc., and such sugars A combination of basic changes. See, for example, U.S. Patent 7,560,111 (Kao et al.).

Where the antibody has an Fc region, the oligosaccharide structure can be attached to one or both of the heavy chains of the antibody, for example at residue 299. In one embodiment, G0 is the major oligosaccharide structure and other oligosaccharide structures are found, such as G0-F, G-1, Man5, Man6, G1-1, G1(1-6), G1 (1-3) And G2 is less in the composition.

Unless otherwise indicated, the G1 oligosaccharide structures herein include G1(1-6) and G1(1-3) structures.

For the purposes herein, a sialylated variant is a variant in which the major class of antibodies comprises one or more sialylated carbohydrate moieties attached to one or both of its heavy chains. Sialylated variants can be evaluated by administering a composition that is or not treated with a sialidase (eg, by ion Identification by exchange chromatography, for example as described in WO2009/099829.

A glycosylated variant is one in which a sugar (e.g., glucose) has been covalently attached to, for example, one or two of its light chains. This addition can occur by reaction of glucose with an amino acid residue on the protein (eg, in cell culture media). Glycosylation variants can be identified by mass spectrometric analysis of the reduced antibodies to assess the increase in mass of the heavy or light chain. As explained in WO 2009/099829 (Harris et al.), saccharification variants can also be used Acid chromatography quantitation.

Deaminated amidated antibodies are those in which one or more of the asparagine residues have been derivatized, for example, aspartic acid, amber imine or isoaspartic acid. An example of a deaminated antibody is a pertuzumab variant in which Asn-386 and/or Asn-391 is removed from one or both of the heavy chains of pertuzumab. See, for example, WO2009/099829 (Harris et al.).

Amino terminal leader stretch variant herein refers to one or more amino acid residues having an amino terminal leader sequence at the amine terminus of any one or more of the heavy or light chain of the major class of antibodies. This major class of antibodies. Exemplary amine-based leader region extensions include or consist of three amino acid residues VHS-present on one or both of the antibody variants, designated herein as "VHS-variants." See, U.S. Patent 7,560,111 (Kao et al.).

"C-terminal acyl acid variant" refers to a variant comprising an amino acid (K) residue at the C-terminus of its heavy chain. See U.S. Patent 7,560,111 (Kao et al.).

By "methionine oxidative variant" is meant a variant comprising one or more oxidized methionine residues based thereon, such as oxidized Met-254. See U.S. Patent 7,560,111 (Kao et al.).

The term "cancer" refers to a physiological condition in a mammal that is characterized by the growth of unregulated cells. Examples of the cancer herein include breast cancer (e.g., metastatic breast cancer), gastric cancer, ovarian cancer, primary peritoneal cancer, and fallopian tube cancer. Examples of cancers herein include HER-2 positive cancers and low HER3 cancers.

A cancer or biological sample that exhibits HER performance, amplification, or activation is in a diagnostic test A cancer or biological sample that exhibits (including overexpression) a HER receptor, an amplified HER gene, and/or otherwise confirms activation or phosphorylation of the HER receptor.

"HER-2 positive" cancers include cancer cells with higher than normal levels of HER2. Examples of HER-2 positive cancers include HER-2 positive breast cancer and HER2-positive gastric cancer. Methods for identifying HER2-positive cancer include: assays for measuring HER2 proteins, such as immunohistochemical assays (IHC), assays for measuring HER2 encoding nucleic acids, such as in situ hybridization (ISH), including fluorescence in situ hybridization (FISH; See WO 98/45479, published October 1998) and chromogenic in situ hybridization (CISH; see, for example, Tanner et al, Am. J. Pathol. 157(5): 1467-1472 (2000); Bella et al, J .Clin.Oncol. 26: (May 20 Supplement; Abstract 22147) (2008)), Southern dot method, or polymerase chain reaction (PCR) techniques, such as quantitative real-time PCR (qRT-PCR); shedding antigen ( For example, the HER2 ECD) assay (see, for example, U.S. Patent No. 4,933,294, issued June 12, 1990, and U.S. Patent No. 5,401,638, issued on March 28, 1995); Depending on the situation, HER-2 positive cancers have an immunohistochemistry (IHC) score of 2+ or 3+ and/or in situ hybridization (ISH) amplification ratio. 2.0.

"Low HER3" cancer includes cancer cells with a lower than normal level of HER3. Examples of low HER3 cancer include ovarian cancer, primary peritoneal, and fallopian tube cancer. See, for example, U.S. Patent No. 7,981,418 (Amler et al.). In one embodiment, the low HER3 is based on HER3 mRNA expression levels (concentration ratio equal to or lower than 2.81, as assessed by qRT-PCR on a COBAS z480® instrument).

"Epitope 2C4" is a region in the extracellular domain of HER2 that binds to antibody 2C4. To screen for antibodies that bind substantially to the 2C4 epitope, routine cross-blocking assays can be performed as described in Antibodies, A Laboratory Manual , Cold Spring Harbor Laboratory, Ed Harlow, and David Lane (1988). Preferably, the antibody blocks about 50% or more of the binding of 2C4 to HER2. Alternatively, epitope mapping can be performed to assess whether an antibody binds substantially to the 2C4 epitope of HER2. Epitope 2C4 includes residues from subdomain II (SEQ ID NO: 2) in the extracellular domain of HER2. 2C4 and pertuzumab bind to the extracellular domain of HER2 at the junction of subdomains I, II and III (SEQ ID NOS: 1, 2 and 3, respectively). Franklin et al, Cancer Cell 5:317-328 (2004).

"Treatment" refers to therapeutic treatment and preventive or preventive measures. The individuals in need of treatment include those who already have cancer and those in which the cancer is to be prevented. Therefore, the patient to be treated herein may have been diagnosed with cancer or may be susceptible to or susceptible to cancer.

The term "effective amount" refers to the amount of cancer effective to treat a patient's cancer. An effective amount of the drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down and better stop) the infiltration of the cancer cells into the surrounding organs; inhibit (i.e., slow down and better stop) the tumor metastasis; To some extent, inhibit tumor growth; and/or to some extent alleviate one or more symptoms associated with cancer. Within a certain range, the drug can prevent growth and/or kill existing cancer cells, which may be cytostatic and/or cytotoxic. An effective amount can prolong the progression of free survival (eg, as measured by changes in response to solid tumors, RECIST or CA-125 changes), leading to objective responses (including partial response to PR or complete response to CR), increasing overall survival, and / or improve one or more symptoms of cancer (eg, by FOSI assessment).

A "fixed" or "flat" dose of a therapeutic agent is used herein to refer to a dose administered to a human patient regardless of the patient's weight (WT) or body surface area (BSA). The fixed or flat dose is therefore not provided as a dose of mg/kg or a dose of mg/m ² but as an absolute amount of therapeutic agent.

"Container" means an object that can be used to hold or hold a pharmaceutical composition or composition. Examples of the containers herein include vials, syringes, intravenous bags, and the like.

An "intravenous bag" or "IV bag" is a bag that can hold a solution that can be administered via a patient's vein. In one embodiment, the solution is a salt solution (eg, about 0.9% or about 0.45% NaCl). Optionally, the IV bag is formed from polyolefin or polyvinyl chloride.

A "vial" is a container suitable for holding liquid or lyophilized preparations. In one embodiment In this case, the vial is a single use vial, such as a 20 ml single use vial with a stopper.

The "package insert" is a leaflet that must be placed in the packaging of each prescription drug at the order of the Food and Drug Administration (FDA) or other regulatory agency. Leaflets usually include the trademark of the drug, its generic name and its mechanism of action; indications for its indications, contraindications, warnings, precautions, adverse reactions and dosage forms; and instructions for recommended doses, times and routes of administration.

"Pharmaceutical composition" is a pharmaceutically active drug (eg, pertuzumab and variant forms, as disclosed herein) and one or more "pharmaceutically active forms" that can be safely administered to a human patient. A composition (eg, a buffer, a stabilizer, a tonicity modifier, a preservative, a surfactant, etc.). The compositions can be, for example, liquid or lyophilized.

A "recombinant" protein is a protein that has been prepared by genetically modified host cells, such as Chinese hamster ovary (CHO) host cells.

"Manufacturing scale" refers to the preparation of proteinaceous drugs (eg, antibodies) on a commercial scale, for example, 12,000 liters (L) or more, using commercial methods approved by the FDA or other regulatory agencies.

"Purification" refers to one or more purification steps, such as protein A chromatography, ion exchange chromatography, and the like.

An "isolated" variant refers to a variant that has been isolated from a major class or wild type antibody by one or more purification or analytical procedures. The biological activity and/or potency of the isolated variants can be assessed.

II. Antibody composition

(i) major species of antibodies

The antibody compositions herein include antibodies that bind to HER2 (HER2 antibodies), optionally humanized HER2 antibodies. Humanized antibodies herein can include, for example, non-human hypervariable region residues that are incorporated into the human heavy chain variable domain and can be further included at selected from 69H, 71H, and 73H (utilized in Kabat et al ., Sequences of Proteins of The framework region (FR) at the position of the group consisting of Immunological Interest , 5th edition of the Public Health Service, National Institutes of Health, Bethesda, MD (1991) is replaced. In one embodiment, the humanized antibody comprises an FR substitution at two or all of positions 69H, 71H and 73H.

Exemplary humanized antibodies of interest herein include VH CDR residues: - GFTFTDYTMX (SEQ ID NO: 17) for CDR-H1, wherein X is preferably D or S, such as GFTFTDYTMD (SEQ ID NO: 23) ;-for DVNPNSGGSIYNQRFKG (SEQ ID NO: 18) for CDR-H2; and/or - NLGPSFYFDY (SEQ ID NO: 19) for CDR-H3, optionally including amino acid modifications of their CDR residues, eg These modifications substantially maintain or improve the affinity of the antibody. For example, antibody variants for use in the methods of the invention can have from about 1 to about 7 or about 5 amino acid substitutions in the above variable heavy CDR sequences. Such antibody variants can be mutated by affinity, for example as follows.

For example, in addition to the heavy chain variable domain CDR residues in the previous paragraph, the humanized antibody may also include a VL CDR residue: - KASQDVSIGVA (SEQ ID NO: 20) for CDR-L1; - for CDR- L2 is SASYX ¹ X ² X ³ , wherein X ^{1 is} preferably R or L, X ^{2 is} preferably Y or E, and X ^{3 is} preferably T or S (SEQ ID NO: 21), such as SASYRYT (SEQ ID) NO: 24); and/or - for CDR-L3 is QQYYIYPYT (SEQ ID NO: 22).

Such humanized antibodies optionally comprise an amino acid modification of the above CDR residues, for example, wherein the modifications substantially maintain or improve the affinity of the antibody. For example, an antibody variant of interest can have from about 1 to about 7 or about 5 amino acids in the above variable light chain CDR sequences. Replace. Such antibody variants can be prepared by affinity mutations.

Affinity matured antibodies that bind to HER2 are also contemplated by the present invention. The parent antibody may be a human antibody or a humanized antibody, for example, comprising the variable light chain and/or variable heavy chain sequences of SEQ ID NOS: 7 and 8, respectively (ie, VL and/or pertuzumab). Or VH) antibody. Affinity matured pertuzumab variants preferably have an affinity that is superior to murine 2C4 or pertuzumab (e.g., about 2 or about 4 fold to about 100 fold or about 1000 fold improved affinity, e.g., using HER2 ECD ELISA As assessed) binds to the HER2 receptor. Exemplary variable heavy CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or two or more (eg, two, three, four, five, six) Or a combination of seven such residues). Examples of variable light chain CDR residues for alteration include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97 or two or more (eg, two to three, four a combination of five or up to about 10 of these residues).

Various forms of humanized antibodies or affinity matured antibodies are contemplated. For example, a humanized antibody or an affinity matured antibody. Alternatively, the humanized antibody or affinity matured antibody can be an intact antibody, such as a full IgGl antibody.

Preferably, the HER2 antibody (either or both of the major class of HER2 antibodies and antibody variants thereof) binds to subdomain II of HER2, inhibits HER dimerization, and/or binds more effectively than trastuzumab. An antibody to a heterodimeric binding site of HER2. Preferred examples of the major classes of antibodies herein are the variable light chain and variable heavy chain amino acid sequences comprising SEQ ID No. 3 and 4, and preferably the light chain amine group comprising SEQ ID No. 11 or 15. An acid sequence and an antibody to the heavy chain amino acid sequence of SEQ ID No. 12 or 16.

(ii) unpaired cysteine variants

Examples 1 and 3 herein describe unpaired cysteine variants of pertuzumab. Analytical assays for liberating, characterizing, and quantifying such variants include assays that specifically assess intrachain disulfide bonds (as opposed to interchain disulfide bonds), for example, antibody fragments as in Example 1 herein (eg, Hydrophobic interaction chromatography (HIC) analysis of Fab fragments), HIC of intact antibodies as in Example 1, peptide mapping analysis of differentially labeled antibodies as in Example 3, and/or as in Example 3 and in Zhang et al. Human, Anal. Chem. 84(16): 7112-7123 (2012) by reversed phase high performance liquid chromatography (RP-HPLC).

In general, the predominant form of pertuzumab involves a disulfide bond between Cys23 and Cys88 in both VL domains of its two Fab domains. See Figure 6.

One of the unpaired cysteine variant heterodimeric variants herein lacks a Cys23/Cys88 disulfide bond in only one variable light chain (VL) domain in both Fab regions. See Figure 20(b). This was identified as a major unpaired cysteine variant.

Another unpaired cysteine variant homodimer variant herein lacks a Cys23/Cys88 disulfide bond in both Fab regions. See Figure 20(c).

In one embodiment, the amount of the unpaired cysteine variant (including homodimers and heterodimer variants) in the composition is Approximately 25%, for example, as determined by Fab hydrophobic interaction chromatography (HIC).

In one embodiment, the amount of homodimeric variant in the composition is 4.9%, as determined by the HIC of the intact antibody.

In one embodiment, the amount of heterodimeric variant in the composition is from about 13% to about 18%, for example, as determined by the HIC of the intact antibody.

The composition further comprises one or more other variants as described below, as appropriate.

The invention also relates to an unpaired cysteine variant of an isolated paclizumab, wherein the unpaired cysteine variant comprises one or two light chain variable domains of pertuzumab Cys23/Cys88 in the unpaired cysteine. The isolated, unpaired cysteine variants can include or consist of heterodimeric variants and/or homodimeric variants. Such variants can be isolated using HIC or other purification methods and can be subjected to bioassays, such as potency assays (using HER2-positive breast cancer cells), as shown in Example 1 below.

(iii) afucosylation-free variant

Example 2 and Example 4 herein describe afucosylated variants of pertuzumab, and Demonstrates how ADCC activity is determined based on the percentage of afucosylated pertuzumab in the composition.

In one embodiment, the invention relates to a composition comprising a fucosylation variant of pertuzumab and pertuzumab, wherein the afucosylated variant is greater than 2 % of the composition. See, for example, anti2C4907-2 and Run 1 in Table 9 below.

In an alternate embodiment, the invention relates to a composition comprising a fucosylation variant of pertuzumab and pertuzumab, wherein the afucosylated variant is present in an amount of 0.9 Up to 4.1% of the composition. This amount of afucosylated variant can be quantified, for example, using the CE-LIF assay in the verified Example 4.

Optionally, the composition further comprises an unpaired cysteine variant (heterodimer and/or homodimer, as described in the previous section) and/or other variants as will be described below.

(iv) LMWS and HMWS

The present invention further relates to a low molecular weight species (LMWS) of pertuzumab and/or a high molecular weight species (HMWS) of pertuzumab, either in isolated form or in a variant comprising and The form of the composition of the main class of antibodies. The LMWS and HMWS can be isolated, characterized, and quantified using a variety of techniques including, but not limited to, size exclusion high performance liquid chromatography (SE-HPLC) and/or capillary electrophoresis sodium dodecyl sulfate (CE-SDS).

Using SE-HPLC assays (eg, as in Example 5), the levels of the major classes of pertuzumab and HMWS or LMWS in the composition can be:

Main peak: About 96%, for example, About 96.7%, About 97.3%, for example, About 97.4%.

HMWS: About 2%, for example, About 1.7%; for example, About 1.5%, for example About 1.4%; for example About 0.8%.

LMWS: About 2%, for example, About 1.6%, for example, About 1.2%, for example About 0.6%.

Using the NR-CE-SDS assay (eg as in Example 5), the main species in the composition The content of tocilizumab and HMWS or LMWS can be:

Main peak: About 95%, for example, About 96.0%, for example, About 97.8%

HMWS: About 1%, for example About 0.6%.

LMWS: About 4%, for example About 3.4%.

For example, the content of the main peak or major species pertuzumab (excluding LMWS and HMWS) may be from about 95% to about 99%, for example, from about 96.0% to about 97.8%, as determined by CE-SDS, for example, 95.3% to about 97.3% of the main peak.

Depending on the situation, the LMWS includes or consists of "Peak 6" (see, for example, Example 5) as obtained by NR-CE-SDS. The peak 6 can be determined to be from about 0.9% to about 2.3%, for example from about 2% to about 2.3%, of the composition.

(v) Peak 1 and peak 2 fragments of pertuzumab

The invention further relates to a peak 1 fragment and/or a peak 2 fragment of pertuzumab which is in isolated or isolated form or in the form of a composition comprising the (equal) fragment and a major class of antibodies. Peak 1 and Peak 2 can be used in a variety of techniques including, but not limited to, size exclusion high performance liquid chromatography (SE-HPLC) and/or capillary electrophoresis sodium dodecyl sulfate (CE-SDS) (including R-CE- SDS and NR-CE-SDS) are isolated, characterized and quantified. In one embodiment, peak 1 and peak 2 are separated and/or analyzed by R-CE-SDS, for example as described in Examples 5 and 6 and the corrected peak area (CPA) provides a peak 1 in the composition or Peak 2%.

Using the R-CE-SDS assay (eg, as in Examples 5 and 6), the peak 1 content of the composition is 5% (eg 0.13% to 0.41% CPA) and the peak 2 content of the composition is 1.0% (eg 0.47% to 0.74% CPA).

(vi) Other variants

The compositions of the present invention include other variants of pertuzumab as appropriate, as described in U.S. Patent 7,560,111 (Kao et al.) and/or WO 2009/099829 (Harris et al.).

Examples of such other variations include, but are not limited to, any one or more of the following: Saccharification variants, disulfide reduction variants, non-reducible variants, deamination variants, sialylation variants, VHS-variants, C-terminal lysine variants, methionine oxidation In vivo, afucosylated variants, G1 glycosylation variants, G2 glycosylation variants, and non-glycosylated heavy chain variants.

For example, the composition may include an acidic variant (see WO 2009/099829, Harris et al.), wherein the acidic variants in the composition may include a saccharification variant, a deamination variant, a disulfide bond One of the reduced, sialylated, and non-reducible variants, two, three, four, or five. Preferably, the total amount of all acidic variants in the composition is less than about 25%. In one embodiment, the saccharification variant, deamination variant, disulfide reduction variant, sialylation variant, and non-reducible variant constitute at least about 75 to the acidic variant in the composition 80%.

Acidic variants can be evaluated by a variety of methods, but preferably such methods include one, two, three, four or five of the following: ion exchange chromatography (IEC), wherein prior to the IEC, The composition is treated with sialidase (eg, to assess sialylation variants), reduced CE-SDS (eg, to assess disulfide reduction variants), non-reduced CE-SDS (eg, to assess non- Revertible variant), Acid chromatography (for example to evaluate glycosylation variants) and peptide maps (for example to assess deamination variants).

The composition optionally includes an amine-based terminal leader extension variant. Preferably, the amino terminal leader region extension is on the light chain of the antibody variant (e.g., on one or both of the antibody variants). An antibody variant herein can be extended on any one or more of its heavy or light chain including an amine terminal leader. Preferably, the amine terminal leader region is extended on one or both of the antibody chains. The amine-based terminal leader region extension preferably comprises or consists of VHS- (i.e., VHS-variant). The presence of the amine terminal leader region extension in the composition can be determined by various analytical techniques including, but not limited to, N-terminal sequence analysis, charge heterogeneity assay (eg, cation exchange chromatography or capillary zone electrophoresis), mass spectrometry Wait for testing. The amount of antibody variant in the composition is generally from any assay used to detect the variant. The lower limit of detection of the preferred cation exchange assay is less than the amount of antibody of the major class. Typically, about 20% or less of the antibody molecule in the composition (e.g., from about 1% to about 15%, such as from 5% to about 15%, and preferably from about 8% to about 12%) comprises an amine terminal leader. District extension. These percentages are preferably determined using cation exchange analysis.

Further amino acid sequence changes of the major classes of antibodies and/or variants are contemplated, including but not limited to one or two antibodies comprising a C-terminal lysine residue on their heavy chain (the antibody variant may be about 1% to About 20% of the content is present, an antibody having one or more methionine residues (for example, pertuzumab including oxidized Met-254), and the like.

Moreover, in addition to the afucosylated variants and sialylated variants described above, the major class of antibodies or variants may include other glycosylation variants, non-limiting examples of which include inclusion of G1 attached to its Fc region or An antibody having a G2 oligosaccharide structure, an antibody comprising one or two non-glycosylated heavy chains, and the like.

III. Manufacturing and analytical methods

According to one embodiment of the present invention, there is provided a method of evaluating a pertuzumab composition comprising one, two, three or four of: (1) measuring an unpaired half of the composition a content of a cysteine variant, wherein the unpaired cysteine variant comprises Cys23/Cys88 unpaired cysteine in one or both of the light chain variable domains of pertuzumab; and/or 2) measuring the amount of afucosylated betuzumab in the composition; and/or (3) measuring the content of the low molecular weight species (LMWS) of pertuzumab in the composition, and / or (4) measuring the content of high molecular weight species (HMWS) of pertuzumab in the composition. All four analytical assays were performed on compositions containing pertuzumab and its variants, as appropriate.

The invention also relates to a method of preparing a composition comprising: (1) preparing a composition comprising pertuzumab and one or more variants thereof, and (2) subjecting the composition thus prepared to one or A variety of analytical assays are available to assess the amount of the (etc.) variant therein. The (equal) analytical assay assesses and quantifies the content of any one or more of the following: (i) contains pertitudin An unpaired cysteine variant of Cys23/Cys88 unpaired cysteine resistant to one or both of the light chain variable domains and/or (ii) only one light chain comprising pertuzumab a heterodimeric variant of Cys23/Cys88 unpaired cysteine in the domain and/or (iii) Cys23/Cys88 unpaired cysteamine in two light chain variable domains comprising pertuzumab a homodimer variant of an acid and/or (iv) an afucosylated variant of pertuzumab and/or (v) a high molecular weight species (HMWS) of pertuzumab and/or (vi) a low molecular weight species of palutizumab (LMWS) and/or (vii) a peak 1 fragment of pertuzumab and/or a (viii) peak 2 fragment of pertuzumab. Therefore, one, two, three, four, five, six, seven or eight of the variants can be analyzed.

Analytical assays assess, quantify, or isolate unpaired cysteine variants, including heterodimers and/or homodimer variants, as appropriate. For example, analytical assays can include hydrophobic interaction chromatography (HIC) of antibody fragments (eg, Fab fragments) or intact antibodies (see, eg, Example 1), peptide mapping analysis (see, eg, Example 3), or reversed phase high performance liquid layer Analysis (HPLC) (see, for example, Example 3).

In one embodiment, the content of the unpaired cysteine variant (heterodimer and/or homodimer variant) in the composition is Approximately 25% was determined by Fab hydrophobic interaction chromatography (HIC).

In one embodiment, the amount of homodimeric variant in the composition is 4.9%, as determined by hydrophobic interaction chromatography (HIC) of intact antibodies.

In one embodiment, the amount of heterodimeric variant in the composition is from about 13% to about 18% as determined by hydrophobic interaction chromatography (HIC) of intact antibodies.

Analytical assays are evaluated, quantified, or isolated from afucosylated variants, as appropriate. The amount of afucosylation can be used to determine or quantify the biological activity of the composition, such as ADCC.

Additionally, the method comprises assessing the biological activity of the pertuzumab composition comprising measuring the amount of afucosylated betuzumab variant in the composition to determine the combination Antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and confirmed that the afucosylated betuzumab content is in the range of from about 0.9% to about 4.1%. For example, the method includes measuring the amount of afucosylated betuzumab using capillary electrophoresis-laser induced fluorescence (CE-LIF).

As appropriate, the analytical assay used to assess afucosylation is capillary electrophoresis (CE), which includes capillary electrophoresis-laser induced fluorescence (CE-LIF), see, Examples 2 and 4 below. The afucosylated variant is optionally present in an amount of from about 0.9 to about 4.1% of the composition (e.g., as measured by CE-LIF in Example 4). In one embodiment, the afucosylated variant is present in an amount greater than 2% of the composition (e.g., as measured by CE-LIF in Example 4).

Analytical assays are evaluated, quantified, or isolated from low molecular weight species (LMWS) and/or high molecular weight species (HMWS) of pertuzumab. Exemplary assays include SE-HPLC and/or CE-SDS (see, for example, Example 5 below).

In one embodiment, the assay comprises SE-HPLC (e.g., as in Example 5), and thus the major species of pertuzumab, HMWS or LMWS in the composition being analyzed is determined to be:

Main peak: About 96%, for example, About 96.7%, About 97.3%, for example, About 97.4%.

HMWS: About 2%, for example, About 1.7%; for example, About 1.5%, for example About 1.4%; for example About 0.8%.

LMWS: About 2%, for example, About 1.6%, for example, About 1.2%, for example About 0.6%.

In one embodiment, the assay comprises CE-SDS (eg, as in Example 5), and thus the major species of pertuzumab, HMWS, or LMWS in the analyzed composition is determined to be:

Main peak: About 95%, for example, About 96.0%, for example, About 97.8%

HMWS: About 1%, for example About 0.6%.

LMWS: About 4%, for example About 3.4%.

In one embodiment, the composition is evaluated by NR-CE-SDS and the content of the main peak or major species pertuzumab (excluding LMWS and HMWS) is measured to be from about 95% to about 99%, such as about From 96.0% to about 97.8%, for example from about 95.3% to about 97.3% of the composition thus analyzed.

In one embodiment, the "peak 6" content of the composition is assessed by CE-SDS (see, eg, Example 5) and the peak 6 LMWS content is determined to be from about 0.9% to about 2.3%, such as about 2 % to about 2.3% of the composition thus analyzed.

In one embodiment, the content of peak 1 and/or peak 2 in the composition is assessed by R-CE-SDS (see, eg, Examples 5 and 6), and the content of Peak 1 is determined as The content of 5% (for example, 0.13% to 0.41% CPA) and peak 2 is determined as 1.0% (eg 0.47% to 0.74% CPA).

The method further optionally comprises combining the purified composition with one or more pharmaceutically acceptable excipients to form a pharmaceutical composition. In addition, the pharmaceutical composition can be placed into a container that is packaged with a package insert (e.g., having prescription information instructing its user to use the pharmaceutical composition to treat cancer) to make a finished article.

IV. Pharmaceutical Composition

A pharmaceutical composition comprising pertuzumab and variants thereof is prepared by mixing a composition of the desired purity with an optional pharmaceutically acceptable excipient ( Remington's Pharmaceutical Sciences 16th Edition Osol, A. Editing ( 1980)) prepared for storage, usually in the form of a lyophilized formulation or aqueous solution. Antibody crystals are also contemplated (see U.S. Patent Application 2002/0136719). Pharmaceutically acceptable excipients are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as histidine acetate; antioxidants, including ascorbic acid and methionine; low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acid such as glycine, amidoxime, aspartame , histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbus alcohol; salt-forming counterions such as sodium; metal complexes (e.g. Zn- protein complexes); and / or non-ionic surfactant, such as polysorbate (e.g. polysorbate 20 or 80), PLURONICS ^TM Or polyethylene glycol (PEG).

The lyophilized antibody formulation is described in U.S. Patent No. 6,267,958, U.S. Patent No. 6,685,940, and U.S. Pat. An exemplary trastuzumab pharmaceutical composition is a white to light yellow sterile lyophilized powder for intravenous (IV) administration without preservative, comprising 440 mg trastuzumab, 400 mg alpha, alpha-algae Sugar dihydrate, 9.9 mg L-histidine-HCl, 6.4 mg L-histamine and 1.8 mg polysorbate 20. 20 mL of bacteriostatic water for injection (BWFI) containing 1.1% benzyl alcohol as a preservative Water produced a multi-dose solution containing 21 mg/mL trastuzumab at a pH of about 6.0.

An exemplary pertuzumab pharmaceutical composition for therapeutic use comprises 30 mg/mL pertuzumab, 20 mM histidine acetate, 120 mM sucrose, 0.02% polysorbate 20, pH 6.0. The replacement pertuzumab formulation contained 25 mg/mL pertuzumab, 10 mM histidine-HCl buffer, 240 mM sucrose, 0.02% polysorbate 20, pH 6.0.

The pharmaceutical composition to be administered for in vivo administration must be sterile. This can be easily accomplished by filtration through a sterile filter.

V. Therapeutic applications and uses

The compositions herein can be used to treat cancer, such as HER-2 positive breast cancer, for example, metastatic or locally recurrent unresectable breast cancer, or a new stage IV disease, defined as immunohistochemistry (IHC) 3+ and/or fluorescein. Photoin situ hybridization (FISH) amplification ratio 2.0. Patients in the cohort have not received prior treatment or relapsed after adjuvant therapy, as appropriate, with baseline 50% left ventricular ejection fraction (LVEF), and/or Eastern Cooperative Tumor Performance Status (ECOG PS) with 0 or 1.

In an alternate embodiment, the composition can be used to treat early HER2-positive breast cancer, for example, in combination with trastuzumab and chemotherapy, wherein the chemotherapy comprises an anthracycline-based chemotherapy, or a carboplatin-based chemotherapy. In one embodiment, the chemotherapy comprises an anthracycline-based chemotherapy, including, for example, 5-FU, epirubicin, and cyclophosphamide (FEC). In an alternate embodiment, the chemotherapy comprises carboplatin-based chemotherapy, including, for example, taxanes (eg, docetaxel), carboplatin, and HERCEPTIN®/trastuzumab (eg, TCH regimen). In one embodiment, the composition is administered concurrently with an anthracycline-based chemotherapy or with a carboplatin-based chemotherapy, for example, wherein pertuzumab, trastuzumab, and chemotherapy are administered in a 3-week cycle. With, pertuzumab, trastuzumab, and chemotherapy administration were administered on the first day of each cycle. Early HER2-positive breast cancer treatments contemplated herein include neoadjuvant and adjuvant therapy.

In another embodiment, the composition can be used to treat HER2-positive gastric cancer, optionally with trastuzumab and chemotherapy, such as platinum (eg, cisplatin) and/or fluoropyrimidine (eg, capecitabine) And/or 5-fluorouracil (5-FU) combination.

In an alternate embodiment, the composition can be used to treat HER2-positive breast cancer, optionally in combination with trastuzumab and vinorelbine. The breast cancer according to this embodiment is optionally metastatic or locally advanced. Depending on the condition, the patient has not previously received a systemic non-hormonal anticancer therapy in a metastatic setting.

In another aspect, the composition is for treating a HER2-positive breast cancer in a patient comprising administering the composition, trastuzumab, and an aromatase inhibitor (eg, anastrazole or letrozole) (letrozole)) to the patient. According to this embodiment, the breast cancer is advanced breast cancer, including hormone receptor-positive breast cancer such as estrogen receptor (ER)-positive and/or progesterone receptor (PgR)-positive breast cancer. Depending on the condition, the patient has not previously received a systemic non-hormonal anticancer therapy in a metastatic setting. The method of treatment further includes administering to the patient an induction chemotherapy (eg, including a taxane), as appropriate.

In another aspect, the composition is for treating a low HER3 cancer, such as ovarian cancer, Primary peritoneal or fallopian tube cancer. See, for example, U.S. Patent 7,981,418 (Amler et al.) and U.S. Patent Publication No. US-2006-0013819-A1 (Kelsey, S.).

Such antibodies and chemotherapy treatments are administered to human patients according to known methods. Specific dosing schedules and formulations are described in the examples herein.

According to a particular embodiment of the invention, about 840 mg (loading dose) of pertuzumab is administered followed by about 420 mg (maintenance dose) of pertuzumab in one or more doses. The maintenance dose is preferably administered about every 3 weeks for a total of at least two doses until clinically progressive disease or uncontrolled toxicity, for example, 6 to 20 doses. Longer treatment periods are also envisaged, including more treatment cycles.

According to another specific embodiment, in the case where the cancer is gastric cancer, pertuzumab is administered at a dose of 840 mg for all treatment cycles.

VI. Making items

One embodiment of a manufactured article herein includes a container comprising a composition or pharmaceutical composition herein, such as a vial, syringe, or intravenous (IV) bag. Optionally, the article of manufacture further includes a package insert having information describing how to use the composition according to the previous section of this document.

Further details of the invention are illustrated by the following non-limiting examples. All of the cited disclosures in this specification are expressly incorporated herein by reference.

Example 1 Cyst23/Cys88 unpaired cysteine variant and its characterization of pertuzumab

Pertuzumab is a humanized monoclonal antibody (MAb) based on the human IgG1 (κ) framework. The recombinant antibody is produced by Chinese hamster ovary (CHO) cells and comprises two heavy chains (each having 449 amino acid residues) and two light chains (each having 214 amino acid residues) and a chain Inter and intrachain disulfide bonds. The light and heavy chain sequences of pertuzumab are shown in Figures 3A and 3B, respectively. The calculated molecular mass of intact pertuzumab is 145,197 Da (peptide chain only, free of heavy chain C-terminal amino acid residues).

The CH2 domain of each heavy chain also has a single conserved glycosylation site at Asn299.

The difference between pertuzumab and trastuzumab (HERCEPTIN®) is the complementarity determining region (CDR) of the light chain (12 amino acid differences) and the heavy chain (29 amino acid differences), and their binding Facts to different epitopes on human epidermal growth factor receptor 2 (p185 ^HER2 ). Binding of pertuzumab to the HER2 receptor on human epithelial cells prevents HER2 from forming a complex with other members of the HER receptor family, including EGFR, HER3, HER4, and prevents the formation of HER2 homodimers. By blocking complex formation, pertuzumab inhibits ligand-induced intracellular signaling via two major signaling pathways (mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K)) Signaling, thereby inhibiting cell proliferation and survival, respectively.

This example relates to the recognition and characterization of an unpaired cysteine variant of pertuzumab: the Cys23/Cys88 unpaired cysteine variant comprises one or two of the light chains. Cystamine.

The free sulfhydryl group was measured using an Ellman reagent and showed a reactive free thiol content of 0.1 to 0.3 mol per mol of protein. Hydrophobic interaction chromatography (HIC) analysis and peptide mapping analysis revealed that the unpaired cysteine residues were at Cys23 and Cys88 on one or both of the light chains. Using papain HIC, the level of the Fab variant containing free sulfhydryl groups at the iso-position was found to be 12.7% to 13.5% in the pertuzumab material prepared using the commercial manufacturing method. HIC analysis of intact antibodies indicated that the two major forms were 78% to 85% wild-type pertuzumab and 13.4% to 18.4% pertuzumab heterodimer (unpaired cysts on one arm) Amino acid pair).

Materials and methods

Test Composition : This example describes the characterization of the current pertuzumab reference standard batch anti2C4907-2 and Run 1 (representing Phase III clinical material) and five Phase III/commercial batches (Run 3 to 7). All were prepared using a commercial process on a 12,000 liter (L) scale. It was also compared to the previous reference standard batch of anti2C4-900-1, which represents the clinical material in Phase I/II.

The composition tested was a pharmaceutical batch formulated in a commercial formulation at 30 mg/mL at pH 6.0 in 20 mM L-histamine acetate, 120 mM sucrose, and 0.02% (w/v) polysorbate 20. The batch anti2C4-900-1 was formulated in an early clinical development at 25 mg/mL in 10 mM L-histamine hydrochloride, 240 mM sucrose, and 0.02% (w/v) polysorbate 20.

Non-reducing peptide mapping and mass spectrometry disulfide analysis : In order to denature the pertuzumab under non-reducing conditions and alkylate any buried free mercapto, it will be contained in the formulation buffer of about 0.5. The mg pertuzumab was mixed with a denaturing buffer (composed of 8 M GdHCl, 10 mM N-ethyl maleimide (NEM), 0.1 M sodium acetate, pH 5.0), and then incubated at 37 ° C for 3 hours. The buffer of this solution was replaced with 600 μL of 0.1 M Tris, 1 mM CaCl ₂ , pH 7.0 using a NAP-5 column. Acetonitrile (ACN) was added to each sample to achieve a concentration of 10%. The trypsin digestion reaction was carried out at 37 ° C for 16 hours at a 1:10 (w/w) enzyme to substrate ratio. The resulting peptide was separated by RP-HPLC using the following method for sulfitolysis trypsin.

Sulfite trypsin peptide map : In order to generate a map of the pertuzumab peptide, the protein was digested by trypsin after reduction and cleavage of the cysteine residue. An aliquot (1 mg) of pertuzumab was added to 360 mM Tris-HCl pH 8.6, 6 M guanidine hydrochloride (GdHCl), 2 mM ethylenediaminetetraacetic acid (EDTA), 13 mM sodium sulfite, and 38 mM sodium tetrathionate. Reduction and sulfite cleavage of cysteine residues. The samples were incubated at 37 ° C for 20 minutes. The sulfite sample was loaded onto a PD-10 column and eluted with 10 mM Tris, 0.1 mM CaCl ₂ , pH 8.3. After buffer exchange, 20 μL of 10% octyl-B-glucoside solution and 20 μL of 1 mg/mL trypsin were added. The samples were incubated at 37 ° C for 5 hours. The digestion reaction was stopped with 25 μL of 10% trifluoroacetic acid (TFA). The resulting peptide was separated by RP-HPLC using a Zorbax 300SB-C8 column (4.6 mm x 150 mm). The peptides are held under initial conditions for 5 minutes, from 0% to 17% solvent B in 57 minutes, 149 minutes to 32% solvent B, 162 minutes to 45% solvent B, and at 173 minutes to 95 minutes. % Solvent B is separated by a linear gradient. At 179 minutes, the column was reconditioned at 100% solvent A for 25 minutes with a total run time of 204 minutes. Solvent A consisted of an aqueous solution of 0.1% TFA and solvent B consisted of 0.08% TFA in acetonitrile. The column was maintained at 37 ° C and eluted at a flow rate of 0.5 mL / min. The elution profile was monitored at 214 nm and 280 nm. The tryptic peptide mass-based separation by liquid chromatography of the digestion mixture - mass spectrometry (LC-MS) analysis using a mass spectrometer LTQ ORBITRAP ^TM measured.

Free thiol content by Elman analysis : Pertuzumab sample buffer was exchanged into reaction buffer (100 mM potassium phosphate, 1 mM EDTA, 8 M urea, pH 8) and adjusted to result in a free thiol concentration The concentration within the range of the standard curve. A solution of dithionitrobenzene (DTNB) (10 mM) and a standard curve of cysteine (8 points between 0 and 100 μM) were prepared in reaction buffer. In a 96-well plate, 165 μL of sample or standard was added to the three replicate wells. The reaction was started by adding 10 μL of DTNB, followed by incubation for 30 minutes. After incubation, the absorbance was measured at 412 nm using a SPECTRAMAX M ² ® plate reader. The linear equation obtained from the standard curve was used to calculate the concentration of free thiol. The concentration of the protein was determined using the absorbance at 280 nm obtained by a spectrophotometer. The free thiol was recorded as the number of free thiol moins per motraptuzumab.

Papain HIC : For papain-digested pertuzumab samples, these samples were digested with papain after removal of the C-terminal lysine using carboxypeptidase B (CpB). The Fab and Fc domains were isolated by HIC using a polyacrylic acid hydrazine column (4.6 mm x 100 mm, 1500 Å, 3 μm). Solvent A consisted of 1.6 M ammonium sulfate, 20 mM potassium phosphate, pH 6.05 and solvent B consisted of 20 mM potassium phosphate, pH 6.05. The analyte was separated from 0% to 18% solvent B from 3 to 6 minutes at a gradient of 21 minutes to 24% solvent B. The column was maintained at 25 ° C at a flow rate of 0.8 mL/min. The elution profile was monitored at 280 nm.

HIC of intact antibody : A complete pertuzumab sample was isolated by HIC using a polyacrylic acid guanidine column (9.4 mm x 100 mm, 1500 Å, 3 μm). Solvent A consisted of 1.0 M ammonium sulfate, 20 mM potassium phosphate, pH 6.05 and solvent B consisted of 20 mM potassium phosphate, pH 6.05. The analyte was separated isocratically with 12% solvent B for 25 minutes. The column was maintained at 30 ° C at a flow rate of 2 mL/min. The elution profile was monitored at 280 nm.

SDS-PAGE using peptide mass fingerprinting : Both reduced and non-reduced pertuzumab samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For the non-reduced sample, the sample (5 μg) was denatured with iodoacetamide by heating at 60 ± 2 ° C for 5 to 10 minutes in the presence of SDS-PAGE sample buffer. The sample was reduced in the presence of 80 mM dithiothreitol (DTT) for 15 to 20 minutes at 60 °C ± 2 °C. Samples were separated on denaturing 4-20% gradient polyacrylamide gel and stained with SYPRO ^TM Ruby dyes to obtain the protein bands FIG. And samples with pertuzumab, and the molecular weight standard ruby dye SYPRO ^TM sensitivity standard (2ng / strip and 8ng / strip bovine serum albumin (BSA)) is included in the gel.

Peptide mass fingerprinting is an analytical technique for protein recognition. The gel was loaded with 10 μg of the commercially available reference standard batch anti2C4907-2 and Run 5. All bands separated by SDS-PAGE were cleaved by trypsin to peptide. Using matrix-assisted laser desorption BRUKER ^TM / ionization - time of flight mass spectrometry (MALDI-TOF MS), accurate measurement of the absolute mass of the peptide. The peptide mass list is used to identify proteins by searching for protein sequences. All observed bands of both non-reduced and reduced pertuzumab were identified by peptide mass fingerprinting.

Efficacy by bioassay : The efficacy of the pertuzumab was evaluated by measuring its ability to inhibit the proliferation of human breast cancer cell lines expressing HER2. In a typical assay, 96-well microtiter plates were seeded with breast cancer cells and cultured in a humidified incubator. After incubation, the medium is removed and different concentrations of pertuzumab reference standards, assay controls, and samples are added to the plate. The plate is then incubated and the relative number of viable cells is quantified indirectly using the redox dye ALAMARBLUE®. Fluorescence was measured using excitation at 530 nm and emission at 590 nm. ALAMARBLUE® is blue and non-fluorescent in its oxidized state, but is reduced to a pink form by the intracellular environment of the cell, which is strongly fluorescent (Page et al., Int. J. Oncol. 3: 473- 476 (1993)). The change in color and fluorescence is proportional to the number of living cells. The results expressed as relative fluorescence units (RFU) are plotted against the pertuzumab concentration, and the parallel line protocol was used to estimate the antiproliferative activity of the pertuzumab sample relative to the reference standard.

result

Disulfide bond partitioning : There are 32 cysteines in pertuzumab, forming 16 disulfide bonds, four of which are interchain and 12 are intrachain links. However, due to the multimeric nature of the molecule, there are only nine different disulfide bonds. The native protein is digested with trypsin to achieve release of all disulfide-linked peptides. A chromatogram of the pastuzumab batch is shown in Figure 7. Reversed-phase LC-MS analysis of the commercially available reference standard batch of digest of anti2C4907-2 yielded all expected disulfide-linked peptide pairs (Table 2).

Note: The equal sign (=) indicates a disulfide bond.

H = heavy chain; L = light chain; LC-MS = high performance liquid chromatography mass spectrometry, T = tryptic peptide.

^a Refer to Figures 9 and 10.

^b Single isotope mass (MH ⁺ ).

^c Inferred disulfide bond. The T19H dimer distribution does not include the verification of Cys228=Cys228 and Cys231=Cys231 disulfide bonds.

^d The presence of this pair of disulfide linkages has been confirmed by the use of the alternative enzyme Lys-C which does not cleave T19L and T20L.

The identified peptide was further confirmed by recognition of the expected peptide from the peptide upon reduction of the disulfide bond (Fig. 8, enlarged view in Fig. 9 and Fig. 10). The dimer of the heavy chain peptide T19H (T19H=T19H) was identified as containing two disulfide bonds; it was concluded that Cys228=Cys228 and Cys231=Cys231 were recognized. A pair of disulfide bonds were detected by LC-MS, T18H = T20L, but the elution was close to the void volume and was not recognized as a distinct peak on the ultraviolet (UV) chromatogram. The presence of this disulfide pair was further confirmed by LC-MS analysis of Lys-C digest, where peptide T18H = T19L-T20L was observed. No unexpected connections were found. As discussed below, a disulfide bond is partially unpaired.

Free thiol analysis : All cysteine residues in the properly folded pertuzumab should be involved in disulfide bonds. Determination of reactive unmodified (free) thiols using the Elman test (Ellman, G. Arch . Biochem . Biophys. 82: 70-77 (1959)), a method for measuring the free sulfhydryl content of peptides and proteins Whether it is present in pertuzumab. The free thiol (unpaired cysteine residues) content of all materials was evaluated and the results are summarized in Table 3.

Note: Free thiol levels were determined by the Elman assay in the presence of 8 M urea.

About 0.1 to 0.3 moles of free thiol/morpazumuzumab was observed in all batches analyzed. In the absence of 8 M urea, the free thiol level in all tested materials was below the limit of quantitation (QL; about 0.1 mol free thiol/mol protein), indicating the presence of the pertuzumab molecule The free thiol (i.e., unpaired cysteine) is buried and cannot be brought close to the Elman reagent under non-denaturing conditions.

After CpB and papain digestion, the pertuzumab material was analyzed by HIC, showing an additional peak between the Fc and Fab peaks, which was identified as a Fab change containing unpaired cysteine residues at Cys23 and Cys88. Body (Figures 11 and 12, labeled as free thiol Fab). This recognition was confirmed by LC-MS tryptic peptide mapping, in which the sample was denatured in the presence of NEM prior to reduction and trypsinization. The extent of free thiol Fab variants of all the pertuzumab batches was measured using the papain HIC method and was found to be consistent with the current method (Table 4).

Note: The unpaired cysteine Fab peak % is obtained by dividing the area of the unpaired cysteine Fab peak by the peak area of the unpaired cysteine Fab + Fab.

* Calculations are as follows.

The value of the material prepared by the commercial method was 12.7% to 13.5% by the papain HIC assay, while the value of the reference standard batch 2C4-900-1 (I/II phase) was slightly lower. 9.4%.

% conversion of Fab variants from papain HIC to estimated intact antibody variant % : The relative amount of Fab fragment containing unpaired cysteine can be used to calculate a heterodimer of an unpaired cysteine variant or Relative distribution of homodimeric forms. If the papain HIC assay shows that 10% (or x%) of the Fab fragment from pertuzumab contains unpaired cysteine at Cys23/Cys88, then 10 should contain unpaired cysteine. The Fab fragment is released from every 50 pertuzumab molecules because 100 Fab fragments should be produced by papain digestion of 50 antibodies. Assuming that these 10 Fab fragments are from 10 different pertuzumab molecules, the relative amount of pertuzumab containing 1 Fab with Cys23/Cys88 unpaired cysteine is about 20%. That is, there are 10 (or 2x%) of every 50 pertuzumab molecules. More precisely, if the probability of a pertuzumab with two Fabs containing Cys23/Cys88 unpaired cysteine is considered, the pertulinum heterodimer unpaired cysteine variant The relative amount should be 2 x 10% x 90% = 18% (or 2 x x% x [100-x]%). In addition, the relative amount of paclizumab homodimer unpaired cysteine variant should be 10% x 10% = 1% (or x% x x%). In this case, the relative amount of pertuzumab comprising two Fabs that do not contain unpaired cysteine on either Fab should be 90% x 90% = 81% (or [100-x]% ×[100-x]%).

In addition, wild-type homodimers (without unpaired cysteine) and heterodimers (containing unpaired cysteine on one Fab) can be directly quantified by HIC. The HIC of the intact antibody divides pertuzumab into two major peaks (Figure 13), which are identified as wild-type homodimers (without free thiols) by LC-MS tryptic peptide mapping and Heterodimer (having a free thiol pair on one Fab). The smaller anterior shoulder was also collected and characterized by papain HIC as the major homodimer (free thiol pair on both Fabs, approximately 40%) and patozumab with Fc oxidation. Using the HIC of the intact antibody, it is estimated that for the materials prepared using the current method, the pertuzumab contains about 17% to 18% of the heterodimer, and for the material prepared using the Phase I/II method, contains 13% ( table 5). Not limited to any one reason It is possible that the increase in unpaired cysteine variants prepared by commercial methods (relative to the Phase I/II method) may result from the protein (ie VL domain) folding rate occurring compared to thiol oxidation. The rate of (disulfide bond formation) is faster, thereby capturing free cysteine in the variant.

Note: The percentage of relative peaks is obtained by dividing the individual peak area by the total peak area of all three peaks.

Since the unpaired cysteine acid pair at Cys23/Cys88 on the light chain of pertuzumab was observed by HIC, the purified fraction of each unpaired cysteine variant was tested in an anti-proliferation assay. . Fabs containing unpaired cysteine were purified and estimated to have reducing potency (estimated potency ~50% relative to the original Fab) (Table 6).

Note: The recorded percentage of activity is relative to the original Fab.

^a Estimate the effectiveness value. The dose response curves are not parallel and the lower flat segments do not converge.

In addition, three intact forms (wild-type homodimers, heterodimers containing free thiols, and homodimers containing unpaired cysteine) are isolated by HIC, and The test was performed using an anti-proliferation potency assay. See Table 7.

Note: The recorded percentage of activity is relative to the pertuzumab reference standard (batch anti2C4907-2).

^a This fraction contains about 40% homodimers containing unpaired cysteine and 60% heterodimer or a mixture of wild-type homodimers.

These data indicate that the unpaired cysteine variant of pertuzumab is present in a composition manufactured on a commercial scale. The HIC method (evaluating Fab fragments or intact antibodies) in this example or the peptide map in Example 3 below is an assay that can be used to assess the presence and amount of unpaired cysteine variants in the pertuzumab composition.

Example 2 Non-fucosylated pertuzumab composition and its characterization

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the cell-mediated immunity. By immunity, effector cells actively lyse target cells that have bound antigen-specific antibodies. Pertuzumab exhibited ADCC activity when tested with HER2 3+ cells, but exhibited minimal activity when observed with HER2 1+ cells (Figure 14).

The level of afucosylation in the Patuxuzumab Phase I and Phase III reference standards was determined by capillary electrophoresis. Compared to the Phase I reference standard, it has a lower G0-F (0.8%), a higher level of afucosylated material in the Patuxuzumab III reference standard (G0-F=2.2%) ) correlated with the observed higher ADCC activity (Figure 15). Also prepared and tested for enzymatic removal Pertuzumab was glycosylated and did not show binding to FcyRIIIa and no ADCC activity (Table 8).

Note: The recorded percentage of activity is relative to the pertuzumab reference standard (batch anti2C4907-2).

ADCC = antibody-dependent cell-mediated cytotoxicity.

These data show that measurement of G0-F (no fucosylation) pertuzumab is an effective means for quantifying ADCC activity of pertuzumab. The experimental system for quantifying afucosylation is as follows.

By capillary electrophoresis (CE) analysis of oligosaccharide: Pertuzumab samples (250 to 500 ug) using protein A-based affinity solid phase extraction technique (PHYTIPS ^TM) and purified by automated liquid handling system. The pertuzumab sample was eluted from Protein A resin using 12 mM hydrochloric acid (pH 2.0) and neutralized with 10 μL of 50 mM sodium succinate. The resulting sample was incubated with 2.5 U/mL PNGase F for 15 hours at 37 °C. The protein was precipitated by centrifugation at 95 ° C for 5 minutes and removed by centrifugation. The supernatant solution containing the released oligosaccharide was dried under vacuum. The released glycans were derivatized with 8-aminoindole-1,2,6-trisulphonic acid (APTS) for 2 hours at 55 ° C in a 15% acetic acid solution containing sodium cyanoborohydride. The analysis of the derivatized polysaccharides was carried out using a capillary electrophoresis (CE) system equipped with a fluorescence detection module, using an argon ion laser (488 nm excitation, 520 nm emission) and an N-CHO coated capillary (50 μm x 50 cm). The running buffer was 40 mM ε-amino-n-hexanoic acid/acetic acid, pH 4.5, 0.2% hydroxypropyl methylcellulose (HPMC). The sample was injected into the capillary by a pressure at 0.5 psi. The separation was carried out at 20 kV and the capillary temperature was maintained at 20 °C.

Pertuzumab containing N-linked oligosaccharides site at Asn299 of the Fc portion of the molecule of the C _H 2 domain. The CE and the APTS marker were used after treatment with PNGase F to determine the relative distribution of neutral oligosaccharides found at this site for each batch.

An electropherogram of CE analysis from the released derivatized oligosaccharides is shown in Fig. 16, wherein Fig. 17 is an enlarged map. The relative amounts of oligosaccharides in the pertuzumab of the analyzed material are summarized in Table 9.

Note: Due to rounding, the total % may not be exactly added to 100%. In addition, small species (<0.5%) may have been included in the total peak area %, but are not recorded in this table.

^a sum of two G1 isomers (see Figure 17).

Oligosaccharides having a G0 structure are the major species (62% to 75%) of all materials. Compared to the current reference standard batch anti2C4907-2 and run 1 (62% to 64%), the G0 glycoform is slightly richer in runs 3 to 7 and the previous reference standard batch 2C4-900-1 (70% to 75%) . The glycoform of G1 is observed as two peaks corresponding to the two isomers having terminal galactose on each of the branches of the two-contact structure. The areas of these two peaks were combined to determine the relative amount of G1 glycoforms. The G1 and G2 glycoforms account for approximately 18% to 29% and 1% to 3%, respectively, of the oligosaccharides released from all materials. Peaks due to other oligosaccharide structures were also observed in the electropherogram (all present at 3% or lower). Such structures include G0-F (G0 lacking nuclear fucose), G0-GlcNAc (G0NA lacking a GlcNAc), Man5 and other small glycoforms (Ma and Nashabeh Anal Chem 71: 5185-92 (1999)). The oligosaccharide structure on pertuzumab is the CHO-derived MAb (Ma and Nashabeh, supra) and the naturally occurring human immunoglobulin (Flynn et al, Mol Immunol 47:2074-82 (2010) )) consistent.

Example 3 Peptide mapping and RP-HPLC for evaluation of unpaired cysteine variants

Materials : Materials and devices used in this experiment included: 3-[N-morpholinyl]propanesulfonic acid (MOPS, Sigma-Aldrich), N-ethyl maleimide (d0-NEM; Thermo Scientific, Rockford) , Il), N-ethyl maleimide (d5-NEM; Cambridge Isotope Laboratory, Andover, MA), L-cysteine (Sigma-Aldrich), trypsin (Promega, Madison, WI), Trifluoroacetic acid (TFA; Fisher, Fair Lawn, NJ), acetonitrile (ACN, Burdick & Jackson, Muskegon, MI). All chemicals and reagents were used as received without further purification.

Differences in antibodies N-ethylmaleimide (NEM) labeling : differential NEM labeling method allows free thiols already present in antibodies to be reduced by d0-NEM labeling and remaining disulfide bridges and labeled with d5-NEM . For the initial d0-NEM label, 100 μL of antibody (3 mg/mL) was gently mixed with 400 μL of a denaturing buffer (7.5 M GdnHCl, pH 5) containing 6.25 mM d0-NEM and incubated at 37 ° C for 2 hours. 20 μL of cysteine (125 mM) was added to the sample and incubated at 37 ° C for 15 minutes to inactivate the remaining d0-NEM. To reduce the remaining disulfide bridge in the antibody, 10 μL of TCEP (0.5 M) was added to the sample and incubated at 37 ° C for 30 minutes. 70 μL of d5-NEM (171 mM) was then added to the sample and incubated at 37 ° C for 2 hours to label the free thiol produced by reduction of the disulfide bridge. A 0.5 mL differential NEM-labeled sample was buffer exchanged using a NAP-5 column and eluted with 0.6 mL of MOPS buffer (20 mM MOPS, 0.5 mM TCEP, pH 7).

Peptide mapping of antibodies: Differential NEM-labeled samples were digested with trypsin at 1:50 (w/w) trypsin:antibody for 2 h at 37 °C. The digestion was stopped with 10% TFA. The trypsinized differential NEM-labeled samples were separated using an Agilent 1200 HPLC system (Agilent, Palo Alto, CA). A Jupiter C18 column (250 x 2 mm, 5 μm) with a 300 Å pore size (Phenomenex, Torrance, CA) was used for chromatographic separation of the samples. The injection volume was 95 μL and the column temperature was 55 °C. The mobile phase A was an aqueous solution of 0.1% TFA and the mobile phase B was 0.08% TFA (vol/vol) contained in 90% acetonitrile. The initial conditions were set to 100% mobile phase A and the first 3 minutes after sample injection was maintained. Mobile phase B increased to 10% over the next 20 minutes, and then increased again to 40% up to 160 minutes and to 100% up to 162 minutes, all changing with a linear gradient. Keep mobile phase B at 100% up to 170 minutes. The column was re-equilibrated with 100% mobile phase A until 195 minutes. The flow rate was maintained at 0.28 mL/min.

The effluent from the HPLC is directly connected to the mass spectrometer LTQ ORBITRAP ^TM electrospray ionization source operating in the positive ion mode. The spray voltage was 4.5 kV and the capillary temperature was 300 °C. The mass spectrometer is operated in a data dependent manner to automatically switch between MS and MS/MS modes. The survey full scan mass spectrum was obtained from m/z 300 to m/z 2000 in FT-Orbitrap with a resolution of R=60,000 set at m/z 400. The five maximum intensity ions were fragmented using a collision induced dissociation (CID) in a linear ion trap with a normalized collision energy of 35% and an activation time of 30 ms and an isolated width of 2.5 m/z units. The Dynamic Exclusion (DE) function is enabled to reduce data redundancy and allows the selection of low-intensity ions for data-dependent MS/MS scanning. The dynamic exclusion parameters are as follows: 30 second repetition duration, 500 exclusion list size, 90 second exclusion duration, 0.76 low exclusion mass width, 1.56 height exclusion mass width and 2 repetitions. Use XCALIBUR ^TM data analysis software.

The current pertuzumab reference standard batch anti2C4907-2 was analyzed using the method described above. We found that 10.9% of the T2L peptide (produced by trypsin digestion and containing Cys23), and 8.3% of the T7L peptide (produced by trypsin digestion and containing Cys88) were labeled with d0-NEM. Since only the unpaired cysteine was labeled with d0-NEM in this experiment, these results indicate that about 10% of Cys23 and Cys88 in anti2C4907-2 are not disulfide-linked (ie, 10% unpaired half) Cystatic acid variant). A calculation similar to that described above was used to convert the percentage of unpaired cysteine Fab variants to the percentage of intact pairs of cysteine, which is estimated to be 18 of the pertuzumab molecule in anti2C4907-2 % is heterodimer unpaired A cysteine variant, 1% of the pertuzumab molecule is a homodimeric unpaired cysteine variant, and 81% is a wild-type homodimer form (no pair of cysteamines) acid). These results are generally consistent with the HIC analysis of either the Fab fragment or the complete pertuzumab.

Limited endonuclease Lys-C digestion to produce Fab : Fab fragments of MAb A were generated by a limited Lys-C digestion procedure. Briefly, MAb A (1 mg/mL) was mixed with Lys-C enzyme in a ratio of 1:400 in 100 mM Tris (pH 7.6), and then the mixture was incubated at 37 ° C for 30 minutes. The reaction mixture was labeled with NEMin pH 5.5, 350 mM sodium acetate and 8 M guanidine hydrochloride. The digest was analyzed using the RP-HPLC method described below.

RP-HPLC conditions : RP-HPLC analysis was performed on an AGILENT 1200 ^(TM) HPLC system (Palo Alto, CA, USA) equipped with a binary gradient pump, autosampler, thermostat column compartment and diode array detector. The system included a Pursuit 3 diphenyl reversed phase column (150 x 4.6 mm, 3 μm, Varian, Lake Forest, CA, USA) running at 75 ° C and at 1 ml/min. Separation was monitored using absorbance at 280 nm. The mobile phase consisted of an aqueous solution of 0.1% TFA (mobile phase A) and 0.09% TFA (mobile phase B) contained in ACN. The 38 minute method begins with a three minute gradient from 32% to 36% mobile phase B followed by a 18 minute linear gradient to 42% mobile phase B. The column was washed with 95% mobile phase B for 5 minutes and equilibrated with 32% mobile phase B for 10 minutes.

RP-HPLC analysis of the free thiol Fab produced by limited Lys-C digestion (Figure 18) indicated that the free thiol Fab was about 13%, consistent with the HIC in Example 1. The NEM-labeled free thiol Fab became more hydrophobic and thus eluted later than the free thiol Fab (Figure 18) and further confirmed the presence of free thiol. See also Figure 19, in which the peptide map demonstrates the free thiol Fab.

Example 4 Quantification by afucosylation of CE-LIF

This example describes a fully validated capillary electrophoresis-laser induced fluorescence (CE-LIF) assay to quantify afucosylated betuzumab variants. For the party disclosed in Example 2 above Modifications to the method include: no mechanical sample preparation, no protein A purification steps to ensure consistent protein concentration between samples and changes in electrophoresis parameters (buffering vehicle concentration).

In this assay, the pertuzumab sample was diluted to 10 mg/mL with the formulation buffer and buffer exchanged into peptide-N-glycanase F (PNGase F) digestion buffer. The aspartate-linked oligosaccharide is then enzymatically released using PNGase F. The released glycan is then derivatized with 8-aminoindole-1,3,6-trisulphonic acid (APTS, a negatively charged fluorophore). APTS provides three negative charges for all glycans, allowing for rapid electrophoretic analysis. The mixture containing the excess derivatizing agent and the APTS-glycan conjugate was analyzed by using CE of the coated capillary which reduced the electroosmotic flow. The separation was monitored using a laser induced fluorescence system using an argon ion laser having an excitation wavelength of 488 nm and an emission bandpass filter of 520 nm.

Using this check, the relationship diagram is shown in FIG. Using a graph (%ADCC=30.133+12.439x, where x=%G0-F) and an ADCC range of 40 to 135%, the final specification of pertuzumab corresponds to 0.9 to 4.1% G0-F.

Thus, using this validated CE-LIF assay, the pertuzumab composition can be evaluated to confirm that the biological activity for ADCC is within the desired range (40 to 135% ADCC activity = 0.9 to 4.1% G0- F).

Example 5 Pertuzumab high molecular weight species (HMWS), low molecular weight species (LMWS) and their characterization

Pertuzumab was analyzed by SE-HPLC and CE-SDS to determine the content of high molecular weight species (HMWS) (typically dimers) and low molecular weight species (LMWS). There was no difference in HMWS after dilution, indicating that the aggregates are non-dissociable. For HMWS quantification, there is good agreement between analytical ultracentrifugation (AUC) and SE-HPLC results, indicating no evidence of size exclusion chromatography loss or underestimation of primary HMWS.

Materials and methods

Tested Pertuzumab Composition : This example describes the current pertuzumab reference standard batch anti2C4907-2 and represents Phase III clinical material run 1 and five Phase III/commercial batches (Runs 3 to 7) The characterizations were all made using a commercial method on a 12,000 liter (L) scale.

Isolation of HMWS : To prepare a representative HMWS for biocharacterization, a batch of Pertuzumab from Run 3 was injected into a preparative HPLC using a preparative SE-HPLC column (TSK G3000SW, 21.5 mm x 600 mm). The system was eluted with an equal gradient mobile phase of 4.5 mL/min as described above. The high molecular weight species are collected fractions and subsequently buffer exchanged into a formulation buffer. Analysis by subsequent SE-HPLC showed that HMWS was 70% pure, and the remainder was mainly the main peak. The main peak was also collected and shown to be 100% pure.

Isolated LMWS : To prepare the isolated LMWS, the batch number from Run 3 was digested with papain and fractional collection was performed using preparative HPLC as above. The main forms were Fc and Fab by complete ESI-MS analysis. Analysis by subsequent SE-HPLC showed that LMWS was 99% pure. The isolated Fab variants were also prepared using papain treatment and collected by preparative IE-HPLC. The Fab variant was shown to be 100% pure by subsequent SE-HPLC analysis.

SE-HPLC : An aliquot of pertuzumab was diluted to 10 mg/mL using a mobile phase (0.2 M potassium phosphate, pH 6.2, 0.25 M potassium chloride). Samples were separated on a homogeneously eluted TSK G3000SW _XL column (7.8 mm x 300 mm). The flow rate was 0.5 mL/min, and the column temperature was room temperature. The elution profile was monitored at 280 nm. With respect to the multi-angle light scattering (the MALS) detection of the use of two sequentially connected inline to WYATT DAWN HELEO ^TM MALS detector (using 658nm laser, detector 17) and a refractive index detector WYATT OPTILAB ^TM Rex The column of the device was used to separate the sample of pertuzumab.

CE-SDS : Each of the pertuzumab batches was derivatized using 5-carboxytetramethylrhodamine amber imidate (a fluorescent dye). After removing the free dye using a NAP-5 column, the non-reduced sample was prepared by adding 40 mM of iodoacetamide and heating at 70 ° C for 5 minutes. For the analysis of the reduced samples, the derivatized pertuzumab was mixed with sodium dodecyl sulfate (SDS) and 1 M DTT to a final concentration of 1% SDS (vol/vol). The sample was then heated at 70 ° C for 20 minutes. The prepared samples were analyzed on a CE system using a 50 μm inner diameter × 31.2 cm fused silica capillary at 20 ° C throughout the analysis. The sample was introduced into the capillary by electric injection at 10 kV for 40 seconds. Separation was carried out in a reverse polarity (negative to positive) mode using a CE-SDS running buffer as a screening medium at a constant voltage of 15 kV. An argon ion laser operating at 488 nm was used for fluorescence excitation, where the resulting emission signal was monitored at 560 nm.

Results and discussion

SE-HPLC provides quantitative information on the molecular size distribution of native proteins. The SE-HPLC diagram of the Pertuzumab batch is shown in Figure 25, and an enlarged view of this figure is shown in Figure 26. The relative peak area distribution of the size exclusion peaks is shown in Table 10.

Note: Due to rounding, the total percentage may not be exactly added to 100%.

HMWS = high molecular weight species; LMWS = low molecular weight species.

^a Value obtained from the reference standard anti2C4907-2.

The proportion of pertuzumab eluted in the main peak was 99% or more in all materials. The content of high molecular weight species (HMWS) ranges from 0.1% to 0.2%, while the low molecular weight species (LMWS) is 0.1%. All batches show similar chromatograms. The purified HMWS fraction, including dimers and aggregates of more carbon numbers, proved to have 46% potency relative to the reference standard batch anti2C4907-2.

SE-HPLC was performed on pure and diluted samples maintained at 30 ° C to check for possible rapid and slow-dissociation aggregation of pertuzumab HMWS due to dilution and/or prolonged exposure at high room temperature. . The HMWS content in the diluted and/or heated reference standard batch anti2C4907-2 was not decreased compared to the control.

Separation of SE-HPLC by MAMS on a reference standard batch of anti2C4907-2 confirmed that the SE-HPLC main peak was a monomer having a molecular weight of about 150 kDa.

The AUC of the settling velocity mode was used to characterize the HMWS present in the pertuzumab sample. Settling velocity is a technique that is independent of size exclusion chromatography and measures the level of HMWS in a sample in the absence of a solid column matrix. AUC was performed on a sample of the pertuzumab with increasing HMWS levels to determine SE-HPLC by comparing the level and type of aggregates determined by sedimentation rate with those determined by SE-HPLC. Is it possible to consistently detect all major pertuzumab HMWS. Five samples ranging from 0.2% to 7.2% total HMWS (determined by SE-HPLC) were characterized by settling velocity and labeled A to E in Tables 11 and 27.

These samples consisted of a representative batch of pertuzumab drugs (labeled A) and four samples enriched in HMWS. Samples enriched in HMWS were selected as representative of extensive degradation mechanisms (light exposure, acidic pH exposure, and purified IE-HPLC alkaline variants).

SE-HPLC showed one primary HMWS peak for samples A, B, C, and E and two HMWS peaks for sample D (Figure 27). For samples A, B, C and E, the AUC showed only one HMWS peak with a sedimentation coefficient of about 9.1 s. In sample D, AUC showed two HMWS peaks with a sedimentation coefficient of about 9.1 S and 10.8 S. The HMWS detected by AUC is consistent with SE-HPLC results; both methods show one major degradation product, as well as a smaller level of larger HMWS in sample D.

A comparison of the quantitative results of these samples from the two methods is shown in Table 11.

Note 1: Sample A consists of a representative batch of pertuzumab drug, sample B consists of a batch of pertuzumab that is exposed to 1.2 mlux of light, and sample C is exposed to light at 3.6 mlux. Pertuzumab batch composition, sample D consisted of a batch of pertuzumab that received an acid treatment at pH 3.2, and sample E consisted of a purified alkaline variant from IE-HPLC.

Note 2: See Figure 27 for the corresponding SE-HPLC chromatogram.

AUC = analytical ultracentrifugation; HMWS = high molecular weight species; RSD = relative standard deviation; SE-HPLC = size exclusion high performance liquid chromatography.

^a Samples A and B have HMWS levels below the quantitative limits of the AUC technique.

For samples C, D and E, the percentage of HMWS measured by the two techniques was in good agreement. The low levels of HMWS present in samples A and B prevented accurate quantification of these species by AUC, which had a quantitative limit of 3.7% (Gabrielson and Arthur, Methods 54: 83-91 (2011)). This is reflected by a significant difference in the percentage of HMWS between SE-HPLC and AUC (Table 11). The correlation of a range of HMWS levels was assessed. The correlation coefficient (Lin, L, Biometrics 45: 255-68 (1989)) was calculated to be 0.97 (N = 5), which indicates good agreement between AUC and SE-HPLC for quantifying HMWS (Figure 28).

These results confirm that SE-HPLC is robust in measuring the HMWS of pertuzumab. SE-HPLC can detect and accurately quantify all HMWS species observed by AUC.

Based on the size non-uniformity, the analysis by SE-HPLC, SDS-PAGE and CE-SDS was consistent between batches. For all batches tested, SE-HPLC assays Similar levels of HMWS (0.1% to 0.2%) and LMWS (0.0% to 0.1%) are shown. By SDS-PAGE analysis, the band diagrams formed by the reduced and non-reduced samples are identical, as is the electropherogram produced by CE-SDS.

In one embodiment, the levels of the main classes of pertuzumab and HMWS variants and LMWS variants as assessed by SE-HPLC are as follows:

96% of the main peak, for example, 96.7% of the main peak, for example, 97.3% of the main peak, for example, 97.4% main peak

2% HMWS, for example, 1.7% HMWS, for example, 1.5% HMWS, for example, 1.4% HMWS, for example 0.8% HMWS.

2% LMWS, for example, 1.6% LMWS, for example, 1.2% LMWS, for example 0.6% LMWS.

Both the pertuzumab HMWS and LMWS fractions purified by SE-HPLC exhibited reduced antiproliferative activity compared to the main peak and control, which was completely effective. All size variants showed comparable HER2 binding activity and FcRn binding activity compared to the control, except for LMWS, which showed lower FcRn binding. Since the LMWS sample contained a 2/3 Fab fragment and a 1/3 Fc fragment, lower anti-proliferative and FcRn binding activities were as expected. HMWS showed higher FcyRIIIa (CD16) V158 binding activity, but lower ADCC activity. LMWS showed lower FcyRIIIa (CD16) V158 binding activity and no ADCC activity was observed for this variant (Table 12).

Note: The percentage of activity recorded is relative to the pertuzumab reference standard (batch anti2C4907-2).

ADCC = antibody-dependent cell-mediated cytotoxicity; HMWS = high molecular weight species; LMWS = low molecular weight species.

^a LMWS sample consists of 2/3 Fab and 1/3 Fc fragments. The values shown reflect the molecular weight based nM/nM adjustment (Fab = 47644 Da, Fc = 52800 Da and full length antibody = 148088 Da).

^The bactuzumab reference standard (batch anti2C4907-2) had a G0-F level of 2.2%, while the control sample had 1.7% G0-F. There was no correlation for the difference in the level of afucosylated materials.

Capillary Electrophoresis of Sodium Dodecyl Sulfate (CE-SDS) : CE-SDS Analysis with Laser-Induced Fluorescence (LIF) Detection is a highly sensitive assay that provides a quantitative assessment of the molecular properties of proteins under denaturing conditions. The method of size distribution. In the CE-SDS analysis of non-reduced samples (Figure 29), pertuzumab migrated to a major peak consisting of 96% to 98% of the total peak area and a smaller peak representing LMWS and HMWS. The HMWS content determined by this technique was 0.6% for all materials tested. The rest of the categories migrate to the LMWS as shown in Figure 30 (enlarged view). The alkylation is used to minimize fragmentation caused by heating the sample (Salas-Solano et al., Anal Chem 78: 6583-6594 (2006)). The relative distribution of the species separated by CE-SDS is listed in Table 13.

Note: Due to rounding, the total % may not be exactly added to 100%.

CE-SDS = capillary electrophoresis of sodium lauryl sulfate.

HMWS = high molecular weight species.

A small difference was observed, with peak 6 increasing from 0.9% of the reference standard batch anti2C4-900-1 (I/II method) to reference standard batch anti2C4907-2, run 1 and run 3 to 7 of 1.7% to 2.3%.

In one embodiment, the main peak of pertuzumab (excluding LMWS and HMWS), as isolated or isolated by NR-CE-SDS, is from about 95% to about 99%, for example, from about 96.0% to about 97.8%. . Depending on the situation, if separated by CE-SDS or isolated, the content of HMWS is 1%, for example The content of 0.6% and LMWS is 4%, for example 3.4%.

Example 6 Detection and quantification of fragmentation of pertuzumab

The purpose of this example was to evaluate size exclusion chromatography (SE-HPLC) for the detection of pertuzumab fragments, reduction capillary electrophoresis of sodium lauryl sulfate (R-CE-SDS) and non-reducing CE-SDS (NR). -CE-SDS) method.

Materials and methods

The samples evaluated in this study are summarized below. These include samples of pertuzumab that have been subjected to various stress conditions that may result in more fragmentation.

̇Reference standard (2C4907-2)

̇ Thermal stress (42 days, 40 ° C)

Sulfonic acid treatment (pH 3.2, 1 day, 40 ° C)

̇ Accelerated stability (30 days, at 40 ° C, then stored at about 5 ° C)

̇ Real-time drug (DP) stability (T=0 and T=548 days, and stored at about 5 °C) and the corresponding drug substance (DS)

SE-HPLC was performed as described in Example 5 above with the following recordable values: LMWS, main peak, HMWS, and all other significant peaks above the limit of quantitation (LOQ).

Reduction of CE-SDS (R-CE-SDS) according to Example 5 above, with recordable value: peak 1. LC, Peak 2, Peak 3, NGHC, HC, Peak 5, Inc. Red., and all other significant peaks above LOQ.

Non-reducing CE-SDS (NR-CE-CDS) was performed according to Example 5 above, wherein sample preparation did not include an antibody reduction step to allow for non-reduction analysis of dithiothreitol (DTT) by elimination from the SDS complexing step.

The qualitative results obtained by SE-HPLC, NR-CE-SDS and R-CE-SDS are shown in Figures 31A to B, 32A to B and 33A to B, respectively, and are shown in Tables 14, 15 and Table 16.

Peak identification is based on Hunt & Nashabeh Analytical Chemistry 71: 2390-2397 (1999) and Ma & Nashabeh Chromatographia Supplement 53: S75-S89 (2001). For NR-CE-SDS analysis, the small peak after peak 2 is usually listed as part of peak 2 in the data report. In this study, this small peak was separately reported as peak 2a to distinguish light chain (LC) fragments.

LC = light chain, HC = heavy chain, L = light, H = heavy

NGHC=non-glycosylated heavy chain, HC=heavy chain, Inc.Red.=incomplete reduction

Data Evaluation : Compare the peak area percentage of the relevant fragment (or corrected peak area percentage, %CPA, for CE-SDS) to determine if any CE-SDS method provides non-redundant information compared to SE-HPLC. Related fragments include peaks with unknown structures, or cleavage-derived products known to contain polypeptide chains. These fragments differ from the heavy and/or light chain fragments present in the antibody product and which are normally observed by CE-SDS to dissociate non-disulfide linkages. Fragment peaks must be resolved from other peaks for sensitive detection and accurate quantification.

Ability to detect small fragments : Small fragments were observed in R-CE-SDS and NR-CE-SDS analyses and were named Peak 1 in both assays. These peaks retain the same general shape and migration time in both assays and increase in a similar manner under stress conditions. Therefore, peak 1 is considered to contain the same species in both assays. As noted in Table 17, two CE-SDS assays are capable of detecting small fragments.

The ability to detect fragments produced by acid hydrolysis (acidic clamps) : long-term exposure to acidic conditions can produce fragments, particularly in the Asp-Pro sequence (pertuzumab heavy chain residues 272 to 273), such as by Supported by mass spectrometry of the acid treated sample, it showed masses at 29039 Da (HC 1 to 272) and 21513 Da (HC 273 to 448 with G0 sugar). The theoretical masses of these forms are 29031Da and 21510Da, respectively. Based on the expected migration times for these forms, the corresponding peaks can be clearly seen in the reduced CE-SDS analysis of acid-treated samples (Peak 2, Figure 34), but at much lower than in the non-reduction assay. The level (lower signal) is detected. It can be speculated that in the NR-CE-SDS assay, the peak 2 fragment was probably disulfide-linked and therefore not detected. Since the level of peak 2 detected by R-CE-SDS in the acid-treated sample (5.58%) is higher than the total LMWS (0.26%) of the sample detected by SE-HPLC, it can be concluded SE-HPLC is not sufficient to detect these forms. Thus, the reduced CE-SDS assay is the only assay presented herein capable of detecting fragmentation due to acid hydrolysis, as described in Table 17.

The ability to detect Fab/DesFab fragments : a good linear relationship between LMWS detected by SE-HPLC and peak 3 from the non-reduced CE-SDS assay (r ² =0.97) (Figure 35). LMWS was identified as containing Fab fragments by studies co-eluting with alcohol-producing Fabs. Similarly, peaks 3 and 5 in the NR-CE-SDS assay were identified by a study of co-migration with enzymatically produced Fab and DesFab (Ma & Nashabeh, supra). The desFab peak results from the production of the heavy chain cleavage of the Fab form and is therefore considered to be equimolar relative to the Fab fragment (corresponding to a mass ratio of 2:1) and, therefore, information of this form can also be obtained by SE- HPLC was obtained indirectly as described in Table 17.

Reduction of CE-SDS peak 3 : R-CE-SDS peak 3 was unique to pertuzumab and was not observed in CE-SDS analysis of other antibodies. The extended characterization results support the conclusion that peak 3 is not a product variant or impurity, but rather a method specific to the pertuzumab-induced artifact consisting of a dissociable form of the LC-LC dimer. A variety of techniques are employed to characterize peak 3.

Peak 3 was observed by R-CE-SDS with UV and LIF detection, indicating that it is not a dye label or sample preparation artifact.

The purified pertuzumab light chain fraction produced a peak 3 with an apparent MW about 2 times the theoretical size of LC when analyzed by R-CE-SDS.

Peak 3 was not observed when the electrophoresis conditions included higher capillary temperatures, and no other co-migrating fragments were observed under these conditions.

Studies involving single amino acid mutations have identified three amino acid residues in LC CDR1 and CDR2 that are associated with LC-LC dimer formation. When either of these three residues is replaced with another amino acid, peak 3 is completely dissociated and can no longer be observed by reducing CE-SDS.

SDS-PAGE analysis combined with MALDI-TOF protein mass fingerprinting (PMF) confirmed that host-free cell proteins were present in pertuzumab, nor were similar bands detected at levels observed by CE-SDS.

Collectively, these results support the identification of peak 3 as a method-induced palutizumab-specific LC-LC dimer.

discuss

An evaluation of the information obtained from the study indicates that:

(1) In terms of detection fragments, NR-CE-SDS does provide non-redundant information compared to SE-HPLC

(2) Non-redundant fragmentation information obtained by the NR-CE-SDS method (compared to SE-HPLC) can also be obtained using the R-CE-SDS method.

As shown in Table 16, the reduction assay detects cleavage products resulting from low pH exposure that may occur during manufacture of the drug substance. Table 18 contains reference standards for pertuzumab, Phase III material (n=3) and values obtained by reduction of CE-SDS peaks 1 and 2 using batches prepared by commercial manufacturing methods (n=39). The 95/99 tolerance interval (TI) for Peak 1 and Peak 2 has been calculated using the k value of 3.2 and is given in Table 18. The 95/99 tolerance interval for peak 1 is 0.0 to 0.4% CPA. The 95/99 tolerance interval for Peak 2 is 0.3 to 0.9% CPA.

This article selects the peak 1 when the drug is released. 0.5% and peak 2 1.0% of the final acceptance criteria.

There is no expected change in DS stability due to cryopreservation of the pertuzumab API. Furthermore, no significant changes were observed for any of these named species based on the R-CE-SDS data (Table 19) of the drug product obtained at T=0 and T=548d.

NGHC=non-glycosylated heavy chain, HC=heavy chain, Inc.Red.=incomplete reduction

<110> American Merchants Nandeke Company Lin A Jie Na Luo Yongxiang Gao Yonghua Chapter

<120> Pertuzumab variant (PERTUZUMAB) and its evaluation

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<210> 23

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Sequences are synthesized.

<400> 23

<210> 24

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Sequences are synthesized.

<400> 24

Claims (13)

A composition comprising Pertuzumab and an unpaired cysteine variant thereof, wherein the unpaired cysteine variant comprises two light chain variable domains of pertuzumab Cys23 and Cys88 and Cys23/Cys88 in one or two of the light chain variable domains are unpaired with cysteine. The composition of claim 1, wherein the unpaired cysteine variant is a heterodimer of Cys23/Cys88 unpaired cysteine in only one light chain variable domain comprising pertuzumab Variants. The composition of claim 1, wherein the unpaired cysteine variant is a homodimer of Cys23/Cys88 unpaired cysteine comprising two light chain variable domains of pertuzumab Variants. The composition of any one of claims 1 to 3, wherein the pertuzumab and the unpaired cysteine variant each comprise a variable light and variable weight of SEQ ID NOS. 7 and 8, respectively. Amino acid sequence. The composition of any one of claims 1 to 3, wherein the pertuzumab and the unpaired cysteine variant each comprise the light chain amino acid sequence of SEQ ID No. 11 or 15 and the SEQ ID The heavy chain amino acid sequence of No. 12 or 16. The composition of any one of claims 1 to 3, further comprising one or more other variants of pertuzumab, wherein the other variant systems are selected from the group consisting of: no fucosylation Body, low molecular weight species (LMWS), high molecular weight species (HMWS), glycosylation variants, disulfide bond reduction variants, non-reducible variants, deamination variants, sialylation variants, VHS-variants C-terminal amido acid variants, methionine oxidative variants, G1 glycosylation variants, G2 glycosylation variants, and non-glycosylated heavy chain variants. The composition of any one of claims 1 to 3, wherein the content of the unpaired cysteine variant in the composition is 25%, as measured by Fab hydrophobic interaction chromatography (HIC). The composition of claim 3, wherein the content of the homodimer variant in the composition is 4.9%, as measured by hydrophobic interaction chromatography (HIC) of intact antibodies. The composition of claim 2, wherein the heterodimeric variant is present in the composition in an amount from 13% to 18% as measured by hydrophobic interaction chromatography (HIC) of intact antibodies. A pharmaceutical composition comprising the composition of any one of claims 1 to 9 and one or more pharmaceutically acceptable excipients. An article of manufacture comprising a container having the pharmaceutical composition of claim 10 therein and a package insert having information indicating that the user is using the pharmaceutical composition to treat a cancer patient. The article of claim 11, wherein the cancer is breast cancer, gastric cancer, ovarian cancer, HER2-positive cancer or low HER3 cancer. An isolated pertuzumab variant, wherein the isolated variant comprises: (a) an unpaired cysteine variant of pertuzumab, wherein the variant comprises Patuxin Cys23 and Cys88 in the two light chain variable domains of the monoclonal antibody and heterodimeric variants of Cys23/Cys88 unpaired cysteine in only one of the light chain variable domains; or (b) An unpaired cysteine variant of benizumab, wherein the variant is Cys23 and Cys88 in two light chain variable domains comprising patozumab and Cys23/ in its two light chain variable domains Cys88 is a homodimer variant of the unpaired cysteine.